FACT SHEET Acrylfentanyl - VAD
33
Science at the service of Public health, Food chain safety and Environment. FACT SHEET Acrylfentanyl May 2017 For more information, please contact: Dr. P. Blanckaert Coordinator Belgian Early Warning System Drugs Scientific Institute of Public Health National Focal Point on Drugs Jyliette Wytsmanstraat 14 B-1050 Brussels, Belgium Tel : 02/642 5408 [email protected]
Transcript of FACT SHEET Acrylfentanyl - VAD
Science at the service of Public health, Food chain safety and Environment.
FACT SHEET
Acrylfentanyl
May 2017
For more information, please contact: Dr. P. Blanckaert Coordinator Belgian Early Warning System Drugs Scientific Institute of Public Health National Focal Point on Drugs Jyliette Wytsmanstraat 14 B-1050 Brussels, Belgium Tel : 02/642 5408 [email protected]
The information in this message is exclusively meant for the EWS-network, and was
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the prior permission of the WIV-ISP. The WIV-ISP takes responsibility for the editing of a press release, if considered as necessary in the framework of its mission.
The information contained in this document is also available on the BEWSD-website (with corresponding pdf-files and analytical data).
This part of the website is not accessible for the general public. A login can be requested by contacting [email protected].
© Scientific Institute of Public Health, Brussels 2011
This report may not be reproduced, published or distributed without the consent of the ISP | WIV.
A. General information Recent sample in Belgium In the first half of May 2017, a death was reported in Belgium after the use of acrylfentanyl. The victim, male, presumably consumed a powder containing acrylfentanyl by sniffing, and was found dead at the scene, in the region of Ghent. It remains unclear whether the victim knowingly consumed acrylfentanyl. Several other powders and tablets were found at the scene, one of which was identified as N-ethylhexedrone, a cathinone of the stimulant category. Created July 2016 Updated May 2017 Type Narcotic drugs Group Opioids Name Acryloylfentanyl/acrylfentanyl Nature of substance Acryloylfentanyl is a piperidinamine, which is structurally related to internationally controlled opiod fentanyl (Schedule I of the 1961 United Nations Single Convention On Narcotic Drugs). They differ in the double bond present in the 2- position of the propane attached to the N-phenyl moiety. Acryloylfentanyl is also structurally related to acetylfentanyl, which has been recently subjected to international control (Schedule I, 1961 UN Convention). Because of the structural similarity of acryloylfentanyl and fentanyl, their GC-MS may be similar, with a mass difference of M-2 for the base peak (m/z 243 for acryloylfentanyl). Systematic chemical name N-(1-phenethylpiperidin-4-yl)-N-phenylacrylamide Other names Chemical names: 1-phenethyl-4-N-acryloylanilinopiperidine Other names: acrylfentanyl, ACF
B. Alerts Alerts 23 deaths associated with acrylfentanyl in Sweden – April – August 2016 The EMCDDA has received reports of 23 deaths associated with acryloyl fentanyl. The deaths occurred between April and August 2016 in Sweden. The presence of acryloyl fentanyl was analytically confirmed in all the cases from biological samples taken from the deceased. The decedents were aged 22 to 54 years old (mean 33, median 29); 4 were female, 19 were male. The cause of death is known for 8 cases: acryloyl fentanyl was the cause of death or a contributing factor in all of them; the remaining 15 cases are still under investigation. Acryloyl fentanyl was the only substance consumed in 2 out of 23 deaths. No details are known regarding the source of the substance or the route of administration except for one case where the death occurred after the person consumed acryloyl fentanyl with friends using a nasal spray. In at least 2 cases the deaths were said to have occurred “suddenly”. Reports to EMCDDA Denmark: On 16 May 2017 the Danish FP reported a post-mortem blood and urine sample analysed on 21.01.2017 by the Department of Forensic Medicine, Aarhus University. The concentration of acryloylfentanyl in blood was 0,0072 mg/kg. Estonia: On 17 November 2016 the Estonian FP informed the death cases of 2016: - 1 pure case of acrylfentanyl death; - 2 cases of combination of acrylfentanyl and regular fentanyl deaths. Latvia: On 10 October 2016 the Latvian FP reported a seizure of 0,3699 g beige powder seized on 24/07/2016 by the Police at Riga. Estonia: On 13 September 2016 the Estonian FP reported 6 seizures of powder during Summer 2016 at Tallin. Finland: On 1 September 2016 the Finnish FP reported a seizure of 99 tablets seized on 8.8.2016. The police seized tablets in the Åland island between Sweden and Finland. Sweden: On 31 August 2016 the Swedish FP reported a seizure of 10ml of liquid seized on 19.04.2016 by the Police at Eskilstuna. Sweden: On 22 August 2016 the Swedish FP reported 23 deaths associated with acryloylfentanyl. The substance has been reported as the cause of death in 5 cases. Slovenia: On 1 August 2016 the Slovenian FP reported a sample of 5g light green powder collected on 10.05.2016 by the National Forensic Laboratory. Sample was shipped from China.
C. Pictures Tablet seized in Finland
Capsule seized in Denmark
D. Clinical information Usage This compound has no known uses, and is currently only found as a (legal) fentanyl derivative sold online as a new psychoactive substance. Health risks Acryloylfentanyl is a potent opioid; overdose risks are higher compared to e.g. heroin or morphine. A study by Zhu measured the analgesic activity of derivatives of fentanyl (mouse, intravenous, hot plate test). This study shows that acryloylfentanyl (ED50= 0.082 mg/kg) and fentanyl (ED50=0.062 mg/kg) have high activity as compared to morphine (ED50=13.9 mg/kg). A study by Essawi reports that, at similar doses, acrylfentanyl had longer-lasting antinociceptive effects than fentanyl (mouse, intraperitoneal, hot plate test). Maryanoff et al. measured the inhibitory concentration in vitro (rat brain opioid receptor, naloxone binding) for acryloylfentanyl (IC50= 1.4 nM), fentanyl (IC50= 1.6
nM) and morphine (IC50= 4.2 nM), indicating that acryloylfentanyl has higher affinity to the opioid receptor. Other uses None known.
E. Legal status Currently legal in Belgium. Controlled in Austria, Cyprus, Denmark, Estonia, Finland, Ireland, Latvia, Lithuania, Norway, Poland, Sweden, Turkey, United Kingdom, China.
F. Chemistry Other chemical names and variants 1-phenethyl-4-N-acryloylanilinopiperidine Chemical Abstracts Service (CAS) registry number 79279-03-1 (HCl) Molecular information Molecular structure:
Molecular formula: C22H26N2O Molecular weight: 334.20 g/mol Identification and analytical profile: It has to be noted that the GC-MS EI-spectra of acryloylfentanyl and fentanyl are similar, but with a mass difference of M-2 for the base peak (m/z 243 for acryloylfentanyl). Analytical spectra can be found at the end of this fact sheet, and were provided by the EDND-website of the EMCDDA. These spectra should be considered confidential, and should not be shared without permission.
G. Recent references
Davide Guerrieri, Emma Rapp, Markus Roman, Gunilla Thelander, Robert Kronstrand, Acrylfentanyl: Another new psychoactive drug with fatal consequences, Forensic Science International http://dx.doi.org/10.1016/j.forsciint.2017.05.010
Ujváry I, et al. Acryloylfentanyl, a recently emerged new psychoactive substance: a comprehensive review. Forensic Tox. 2017. doi: http://dx.doi.org/10.1007/s11419-017-0367-8
Watanabe S, et al. In vitro and in vivo metabolite identification studies for the new synthetic opioids acetylfentanyl, acrylfentanyl, furanylfentanyl, and 4-fluoro-isobutyrylfentanyl. AAPS J. 2017. doi: https://dx.doi.org/10.1208/s12248-017-0070-z
Helander A, et al. Intoxications involving acrylfentanyl and other novel designer fentanyls – results from the Swedish STRIDA project. Clin Tox. 2017. doi: http://dx.doi.org/10.1080/15563650.2017.1303141
J. Suzuki, S. El-Haddad, A review: Fentanyl and non-pharmaceutical fentanyls, Drug and Alcohol Dependence 171 (2017) 107–116, http://dx.doi.org/10.1016/j.drugalcdep.2016.11.033
Anders Helander & Matilda Bäckberg (2016): New Psychoactive Substances (NPS) – the Hydra monster of recreational drugs, Clinical Toxicology.
Figure 6: Mass spectrum for precursor (+MS) (top) and product ions (+bbCID) (bottom) for
fentanyl, (C22H28N2O). The product ion at m/z 188.1437 corresponds to C13H18N, which is a well
known fragmentation pathway for fentanyl.
Figure 7: Mass spectrum for precursor (+MS) (top) and product ions (+bbCID) (bottom) for
acrylfentanyl, (C22H28N2O). The product ion at m/z 188.1437 corresponds to C13H18N, which is a
well known fragmentation pathway for fentanyl.
Page 1 af 13
Supporting information for:
Identification of a new psychoactive substance in seized material: The synthetic opioid
N-phenyl-N-[1-(2-phenethyl)piperidin-4-yl]prop-2-enamide (Acrylfentanyl)
Table of contents
Characterisation of acrylfentanyl (seized sample) 2
Acrylfentanyl (seized sample), 1H NMR, 400 MHz (CD3OD) 2
Acrylfentanyl (seized sample), COSY, 400 MHz(CD3OD) 3
Acrylfentanyl (seized sample), MALDI-TOF HRMS 3
Characterisation of fentanyl (standard) 4
Fentanyl (standard), 1H NMR, 400 MHz (CD3OD) 4
Fentanyl (standard), MALDI-TOF HRMS 5
Acrylfentanyl (seized sample) compared to acrylfentanyl (standard), 1H NMR, 600 MHz (CDCl3) 5
Enlargements: Acrylfentanyl (seized sample) compared to acrylfentanyl
(standard), 1H NMR, 600 MHz (CDCl3) 6
Acrylfentanyl (seized sample) compared to acrylfentanyl (standard),
DEPT, 600 MHz (CDCl3) 7
Acrylfentanyl (seized sample), 1H NMR, 600 MHz (DMSO-d6) 7
Acrylfentanyl (seized sample) compared to triethylamine hydrochloride,
DEPT, 600 MHz (DMSO-d6) 8
Acrylfentanyl (seized sample) spiked with triethylamine hydrochloride, 1H NMR, 600 MHz (DMSO-d6) 8
IR spectrum of acrylfentanyl (seized sample) 9
IR spectrum of acrylfentanyl (standard) 9
Instrumentation (LC-MS/MS) for quantification of acrylfentanyl in seized sample 10
Multiple Reaction Monitoring (MRM) parameters 10
LC-MS/MS calibration curve 11
MRM signal for m/z 335 → m/z 188 (acrylfentanyl in seized sample) 11
Qualifying ion transitions (overlaid) for acrylfentanyl in seized sample 12
MRM signal for m/z 343 → m/z 105, internal standard, fentanyl-d5,
spiked to seized sample during sample preparation 12
MRM signal for m/z 335 → m/z 188,
Acrylfentanyl calibrator, 156.25 ng/mL (free base) 13
Qualifying ion transitions (overlaid),
Acrylfentanyl calibrator, 156.25 ng/mL (free base) 13
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Characterisation of acrylfentanyl (seized sample)
The numbering of atoms used for NMR assignments does not follow IUPAC
recommendations and is only used here for clarity (See Fig. S1 for numbering system).
Acrylfentanyl: N-phenyl-N-[1-(2-phenethyl)piperidin-4-yl]prop-2-enamide 1H NMR (400 MHz, CD3OD): δ 7.60-7.52 (m, 3H; Ph), 7.39-7.27 (m, 7H, Ph), 6.31 (dd, J =
17, 2 Hz, 1H, H-1), 5.91 (dd, J = 17, 10.5 Hz, 1H, H-2) 5.59 (dd, J = 10.5, 2 Hz, 1H, H-1’),
4.90 (tt, J = 12, 4 Hz, 1H, H-5),# 3.78 (m, 2H, H-7 and H-7’),* 3.37-3.33 (m, 2H, H-8),# 3.29-
3.22 (m, 2H, H-7 and H-7’),* 3.10-3.05 (m, 2H, H-9), 2.26-2.19 (m, 2H, H-6 and H-6’), 1.86
(qd, J = 8, 4 Hz, H-6 and H-6’)*. # Overlapping with signal from the NMR solvent; *
Overlapping with unidentified impurity.
HRMS (MALDI-TOF) m/z found: 335.2114 [M+H]+; C22H27N2O+ requires M, 335.2118.
Figure S1: Acrylfentanyl (seized sample), 1H NMR (400 MHz) in CD3OD
Overlapping signals from the NMR solvent and impurity triethylamine hydrochloride are
indicated with arrows.
Impurity (Et3N.HCl)
NMR solventresidual signal
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Figure S2: Acrylfentanyl (seized sample), COSY (400 MHz) in CD3OD
Coupling between the CH2- and CH3-groups of triethylamine hydrochloride is observed by
COSY
Figure S3: Acrylfentanyl (seized sample), MALDI-TOF HRMS spectrum DPS2 #1-4 RT: 0.01-0.24 AV: 4 NL: 6.80E6T: FTMS + p MALDI Full lock ms [150.00-600.00]
335 340 345 350 355 360 365 370m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
335.2114
336.2117
357.1924
337.2167 358.1957 373.1664361.2260 369.2312349.2258345.1938 363.2420343.2150 351.1687 355.2150339.1142
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Characterisation of fentanyl (standard)
Fentanyl: N-phenyl-N-[1-(2-phenethyl)piperidin-4-yl]propanamide
The numbering of atoms used for NMR assignments does not follow IUPAC recommendations
and is only used here for clarity (See Fig. S4 for numbering system). 1H NMR (400 MHz, CD3OD): δ 7.57-7.46 (m, 3H, Ph), 7.31-7.23 (m, 4H, Ph), 7.22-7.15 (m,
3H, Ph), 4.62 (tt, J = 12, 4 Hz, 1H, H-5), 3.14-3.02 (m, 2H), 2.83-2.71 (m, 2H), 2.62-2.50 (m,
2H), 2.31-2.15 (m, 2H) (H-6, H-6’, H7, H-7’, H-8 and H-9), 2.01 (q, J = 7.5 Hz, 2H, H-2), 1.88
(d, J = 12.5 Hz, 2H), 1.49 (qd, J = 12.5, 4 Hz, 2H), 1.03 (t, J = 7.5 Hz, 3H, H-1).
HRMS (MALDI-TOF) m/z found: 335.2114 [M+H]+; C22H27N2O+ requires M, 335.2118.
Figure S4: Fentanyl (standard), 1H NMR (400 MHz) in CD3OD
N
N
O
12
56
7
89
3 4
10
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Figure S5: Fentanyl (standard), MALDI-TOF HRMS spectrum
Figure S6: 1H NMR (600 MHz) in CDCl3
Top: Acrylfentanyl (standard); Bottom: Acrylfentanyl (seized sample)
The acrylfentanyl standard conforms to the seized sample, excl. triethylamine hydrochloride
DPS1 #2-4 RT: 0.13-0.25 AV: 3 NL: 7.09E6T: FTMS + p MALDI Full lock ms [150.00-600.00]
340 345 350 355 360 365 370 375m/z
0
5
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45
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55
60
65
70
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100
Rel
ativ
e A
bund
ance
337.2276
359.2086
338.2284
360.2113
375.1825339.2323361.2145353.2217 357.1933 368.4242350.9867340.2353 373.1872347.0517 365.0638345.1918
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Figure S7: 1H NMR (600 MHz) in CDCl3 : Enlargement (4.7–7.7 ppm)
Top: Acrylfentanyl (standard); Bottom: Acrylfentanyl (seized sample)
Figure S8: 1H NMR (600 MHz) in CDCl3 : Enlargement (1.1–3.8 ppm)
Top: Acrylfentanyl (standard); Bottom: Acrylfentanyl (seized sample)
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Figure S9: DEPT (600 MHz) in CDCl3
Top: Acrylfentanyl (standard); Bottom: Acrylfentanyl (seized sample)
Figure S10: Identification of the impurity as triethylamine hydrochloride by 1H NMR (600 MHz) in DMSO-d6. Acrylfentanyl (seized sample),
Peak separation was achieved in DMSO-d6 allowing identification of the impurity.
Page 8 af 13
Figure S11: Identification of the triethylamine hydrochloride impurity by 13C NMR (600 MHz) in DMSO-d6
Top: Acrylfentanyl (seized sample); Bottom: Triethylamine hydrochloride (standard).
Figure S12: Identification of the impurity as triethylamine hydrochloride. Enlargement
of aliphatic region for A) Acrylfentanyl (seized sample); and B) Acrylfentanyl (seized
sample) spiked with triethylamine hydrochloride, 1H NMR (600 MHz) in DMSO-d6
Page 9 af 13
Figure S13: IR spectrum of acrylfentanyl (seized sample)
Figure S14: IR spectrum of acrylfentanyl (standard)
Page 10 af 13
Instrumentation (LC-MS/MS) for quantification of acrylfentanyl
The LC system modules were all from Agilent Technologies (Palo Alto, CA, USA) including
a 1200 binary pump, 1200 SL autosampler and 1200 column department unit. The damper
and mixer were bypassed in order to optimize the pumping system to low dead volume as
described in the Agilent User Manual. Autosampler injection volume was 2 µL. The
analytical column was an Kinetex Biphenyl (Phenomenex, Torrace, CA, USA), 100 × 3 mm,
i.d., packed with 2.6 µm particles. The flow rate was 550 µL/min and the column temperature
was 40 oC. Mobile phase A was 0.1 % formic acid. Mobile phase B was 0.1% formic acid in
methanol. The binary pump gradient started at 2% phase B for 0.5 min and then went up to
95% phase B in 5 min. It was maintained at 95 % phase B for 3 min and then returned to the
initial conditions for equilibration. The total run time was 9 min.
The MS system consisted of an Agilent 6460 triple quadrupole mass spectrometer (Palo Alto,
CA, USA) equipped with a jet stream electrospray ion source operated in positive mode. The
capillary voltage was 3500 V, the nebuliser pressure 25 p.s.i., the gas temperature 350 oC, the
gas flow 8 L/min, the sheath gas temperature 375 oC ion, the sheath gas flow 8.5 L/min, the
nozzle voltage 400 V and the declustering potential 130 V. Data were acquired in dynamic
multiple reaction monitoring mode (dMRM). All calculations were performed used the
MassHunter software (Agilent Technologies).
Table S1: Multiple Reaction Monitoring (MRM) parameters
Compound Transition
type
MRM
transistion
Collision
energy
Fentanyl Target ion 337 → 188 20
Qualifying ion 337 → 105 40
Qualifying ion 337 → 79 50
Acrylfentanyl Target ion 335 → 188 20
Qualifying ion 335 → 105 40
Qualifying ion 335 → 79 50
Fentanyl- d5 Target ion 343 → 105 40
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The calibrators of acrylfentanyl were prepared from a 250 µg/mL aqueous working solution
of acrylfentanyl at six concentration levels: 15.625, 31.25, 62.5, 156.25, 312.5 and 625
ng/mL (free base). The concentration of acrylfentanyl in the seized sample was calculated
from a 6-point calibration curve based on peak area using a linear regression curve fit, not
forced through zero, with no weighting.
A working solution of the seized sample (powder) at concentration 250 ng/mL was used in
the following sample preparation procedure (for sample(s) and calibrators):
100 µL sample was added to 25 µL internal standard solution (fentanyl-d5, 1 µg/mL) and 125
µL water.
The determined acrylfentanyl free base concentration from LC-MS/MS was used to calculate
the mass-% of acrylfentanyl hydrochloride in the seized sample.
Figure S15: LC-MS/MS calibration curve
Figure S16: MRM signal for m/z 335 → m/z 188 (acrylfentanyl in seized sample)
ACRYLFENTANYL - 6 Levels, 6 Levels Used, 6 Points, 6 Points Used, 0 QCs
Relative Concentra
-20 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660
Rel
ativ
e R
espo
nse
1x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
99983033
+ MRM (335.2 -> 188.1) jul-15-2016_ACRYL_11.d
Acquisition Time (m
5 5.05 5.1 5.15 5.2 5.25 5.3 5.35 5.4
Cou
nts
5x10
0
0.5
1
1.5
2
2.5
3
5.103 min.
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Figure S17: Qualifying ion transitions (overlaid) for acrylfentanyl in seized sample
Figure S18: MRM signal for m/z 343 → m/z 105 (internal standard, fentanyl-d5) spiked
to seized sample during sample preparation
Acquisition Time (m
5 5.05 5.1 5.15 5.2 5.25 5.3 5.35 5.4
Rel
ativ
e A
bund
ance
(%
2x10
0
0.2
0.4
0.6
0.8
1
335.2 -> 188.1 , 335.2 -> 105.0 , 335.2 -> 79.0
Ratio = 104.4 (99.3 %)
Ratio = 13.8 (100.3 %)
+ MRM (343.2 -> 105.0) jul-15-2016_ACRYL_11.d
Acquisition Time (m
4.95 5 5.05 5.1 5.15 5.2 5.25 5.3 5.35
Cou
nts
4x10
0
1
2
3
4
5
5.106 min.
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Figure S19: MRM signal for m/z 335 → m/z 188
Acrylfentanyl calibrator, 156.25 ng/mL (free base)
Figure S20: Qualifying ion transitions (overlaid)
Acrylfentanyl calibrator, 156.25 ng/mL (free base)
+ MRM (335.2 -> 188.1) jul-15-2016_ACRYL_3.d
Acquisition Time (m
5 5.05 5.1 5.15 5.2 5.25 5.3 5.35 5.4
Cou
nts
5x10
0
0.5
1
1.5
2
2.5
5.103 min.
Acquisition Time (m
5 5.05 5.1 5.15 5.2 5.25 5.3 5.35 5.4
Rel
ativ
e A
bund
ance
(%
2x10
0
0.2
0.4
0.6
0.8
1
335.2 -> 188.1 , 335.2 -> 105.0 , 335.2 -> 79.0
Ratio = 105.3 (100.2 %)
Ratio = 13.7 (99.3 %)
REVIEW ARTICLE
Acryloylfentanyl, a recently emerged new psychoactive substance:a comprehensive review
Istvan Ujvary1 • Rita Jorge2 • Rachel Christie2 • Thomas Le Ruez2 •
Helgi Valur Danielsson2 • Robert Kronstrand3,4 • Simon Elliott5 • Ana Gallegos2 •
Roumen Sedefov2 • Michael Evans-Brown2
Received: 18 April 2017 / Accepted: 25 April 2017! Japanese Association of Forensic Toxicology and Springer Japan 2017
Abstract N-(1-Phenethylpiperidin-4-yl)-N-phenylacry-lamide, or acryloylfentanyl (acrylfentanyl), is a synthetic
opioid and a close structural analogue of fentanyl, which is
widely used in medicine as an adjunct to general anaesthesiaduring surgery and for pain management. Until recently,
acryloylfentanylwas knownonly from the scientific literature,
but in 2016 this non-controlled substance became available onthe illicit drug market as a powder and nasal spray in Europe
and the USA. By the end of 2016, detection of acryloylfen-
tanyl in six European countries, including 47 deaths associ-ated with the drug, had been reported to the European
Monitoring Centre for Drugs and Drug Addiction
(EMCDDA) through the European Union Early WarningSystem, which is a part of the system designed to identify and
respond to the appearance of new psychoactive substances
that may pose potential public health risks similar to drugscontrolled under theUnitedNations drug control conventions.
Herein we review what is known about this potent narcotic
opioid. In addition to describing its chemical properties andthe synthetic routes, analytical methodologies for the
identification of the substance, as well as the limited infor-mation on the biological properties, including in vitro and
in vivo pharmacological studies with the substance, are
summarised.Analytically confirmed acute intoxications showthat the signs and symptoms of acryloylfentanyl poisoning
correspond to the opioid overdose triad of decreased con-
sciousness, miosis and respiratory depression. Importantly,naloxone works as an antidote in life-threatening poisoning.
The major human urinary metabolites identified in fatal
overdose cases were nor-acryloylfentanyl, as well as mono-and dihydroxylated derivatives and their conjugates.
Keywords Acryloylfentanyl (acrylfentanyl) ! Fentanyl !Fentanils ! New psychoactive substances ! Opioids !Pharmacology and toxicology
Introduction
The elucidation of the chemical structure of morphine byGulland and Robinson in 1925 [1] paved the way for the
development of synthetically more tractable yet potent
analgesics, cough suppressants and antidiarrheal agents,including pethidine, methadone, fentanyl, tramadol,
tapentadol, ketazocine, dextromethorphan and loperamide[2]. However, the idealized goal to find potent opioid
medicines devoid of serious side effects, such as abuse
liability, dependence potential and respiratory depression,has so far eluded researchers.
In the early 1960s, a series of structurally simple yet
highly potent 4-anilinopiperidine-type analgesics weredeveloped in the Research Laboratorium of Dr. Janssen
[3, 4]. Fentanyl (Fig. 1), the first narcotic analgesic medi-
cine belonging to this class, was followed by other struc-turally related substances, the fentanils [5, 6]. Due to the
Electronic supplementary material The online version of thisarticle (doi:10.1007/s11419-017-0367-8) contains supplementarymaterial, which is available to authorized users.
& Istvan [email protected]
1 iKem BT, Buza u. 32, Budapest 1033, Hungary
2 European Monitoring Centre for Drugs and Drug Addiction,Lisbon, Portugal
3 Department of Forensic Genetics and Forensic Toxicology,National Board of Forensic Medicine, Linkoping, Sweden
4 Division of Drug Research, Linkoping University, Linkoping,Sweden
5 Alere Forensics, Malvern, Worcestershire, UK
123
Forensic Toxicol
DOI 10.1007/s11419-017-0367-8
rapid onset and short duration of analgesic action and a
relatively wide therapeutic window, a small number of this
family of compounds, such as fentanyl, alfentanil, sufen-tanil and remifentanil, have become widely used in human
medicine in surgical procedures as adjuncts to anaesthesia
and for the treatment of acute and chronic pain in variousformulations, while some are used in veterinary medicine
as general anaesthetics, for pain management, and, in the
case of carfentanil and thiafentanil, to immobilise largeanimals [7, 8].
Like other opioid analgesics, the analgesic activity of
the fentanils is due to their activation of opioid receptors,and in particular, the l-opioid receptor [9]. Besides their
analgesic properties, a notable feature associated with l-opioid receptor agonists is that they induce dose-dependentrespiratory depression [9–11], which can be life-threaten-
ing. The importance of this effect is reflected in the fact
that most opioid-related deaths are caused by respiratorydepression [12].
Between 1979 and 1988, more than ten fentanils, in-cluding a-methylfentanyl, 3-methylfentanyl and 4-fluoro-
fentanyl, were detected on the illicit opioid market in the
United States, typically sold as heroin or ‘‘synthetic her-oin’’. Together, they were associated with more than 100
overdose deaths during this period [13–15]; later, in the
mid-2000s, outbreaks of poisonings linked to clandestinelyproduced fentanyl also occurred [16–19]. With the excep-
tion of Estonia, which saw an epidemic mainly related to
3-methylfentanyl and fentanyl [20, 21], fentanils causedlimited problems in Europe [22–25]. In the past few years,
there has been a large increase in non-fatal and fatal poi-
sonings in the United States and Canada associated withfentanils that have mostly been sold on the illicit opioid
market: the fentanils are either sold as heroin to unsus-
pecting users, used to cut heroin, or used to make coun-terfeit opioid medicines [26–28]. They have also been sold
as other drugs such as cocaine and benzodiazepines
[29–31].At the same time, Europe has also seen an increase in
the availability of new fentanils. Twenty such substances
have been identified on Europe’s drug market since2012, with eight identified in 2016 alone. Seizures by law
enforcement have also increased, as have poisonings and
deaths (see, for example, [32, 33]). These developments are
part of the broader changes seen on Europe’s drug market
as a result of the appearance of large numbers of new
psychoactive substances over the past decade [34, 35].In Europe, a three-step legal framework of early warn-
ing, risk assessment and control measures allows the
European Union (EU) to rapidly identify and react topublic health threats caused by such substances. The
European Monitoring Centre for Drugs and Drug Addiction
(EMCDDA) is responsible for the first two steps in thesystem, namely operating the EU Early Warning System on
new psychoactive substances with Europol (the EU Police
agency) and conducting risk assessments. As part of thiswork, the EMCDDA monitors more than 620 new psy-
choactive substances that have appeared on Europe’s drug
market over the past 20 years. Data on these substances arecollected and reported by national early warning systems of
the 28 EU member states, Turkey and Norway to the
EMCDDA through the EU Early Warning System as wellas from other sources. Based on data reported through the
EU Early Warning System on acryloylfentanyl, theEMCDDA undertook a detailed investigation on this sub-
stance as part of its early warning and risk assessment
activities [33].Herein we provide a comprehensive and up-to-date
review on acryloylfentanyl, a close structural analogue of
fentanyl, which was first identified on Europe’s drugmarket in 2016 [33] (for the methodology used for the
preparation of the review, see supplementary material).
Following the presentation of the physicochemical prop-erties, the analytical methods and the known synthetic
routes of this new psychoactive substance, and the in vitro
and in vivo pharmacology studies, including mode ofaction and assumed metabolism and toxic effects, are
reviewed and related to those of fentanyl. Finally, serious
adverse events, including symptoms of acute intoxicationand deaths reported within Europe in 2016, are described.
Acryloylfentanyl, as a new psychoactive substance
Acryloylfentanyl is a structurally simple though less stud-ied analogue of fentanyl that was first described in the
scientific literature in the 1980s [36, 37]. In early 2016,
however, the substance was detected on the illicit drug
Fig. 1 Structures of fentanyl,acryloylfentanyl and acryloyl-a-methylfentanyl
Forensic Toxicol
123
market in Denmark and Sweden, and since then, has been
associated with more than 40 deaths in Europe. Acry-loylfentanyl, or ‘‘acryl fentanyl’’, was also identified in the
USA in the fourth quarter of 2016 [38].
Chemically, acryloylfentanyl is an acrylamide deriva-tive of 4-anilinopiperidine and is an unsaturated analogue
of fentanyl (Fig. 1), which is a propionamide and is
included in Schedule I of the United Nations Single Con-vention on Narcotic Drugs of 1961, as amended by the
1972 Protocol. Neither acryloylfentanyl nor any otherunsaturated amides were claimed in the original fentanyl
patent by the Research Laboratorium Dr. C. Janssen [3].
While acryloylfentanyl is new on the international drugmarket, its side-chain methylated homologue, acryloyl-a-methylfentanyl (Fig. 1), was found in seized drugs in the
USA decades ago [13, 39, 40].Apart from acryloylfentanyl, other commonly used
names are acryloyl fentanyl, acrylfentanyl, acryl fentanyl,
and akrylfentanyl (in Sweden). Its systematic (IUPAC)name is N-phenyl-N-[1-(2-phenylethyl)piperidin-4-yl]prop-
2-enamide. Its Chemical Abstract name is N-phenyl-N-[1-
(2-phenylethyl)-4-piperidinyl-2-propenamide. Acryloyl-fentanyl is also known by other names such asN-(1-phenethyl-
piperidin-4-yl)-N-acroylanilinopiperidine, N-(1-phenylethyl-
piperidin-4-yl)-N-phenylacrylamide, or enfentanyl. Its com-mon street names are ‘‘acryloyl-F’’ and ‘‘Acr-F’’. The acronym
‘‘ACF’’ has also been used, which should not to be confused
with ‘‘AF’’, which is one of the street names for acetylfentanyl[32].
Chemical Abstract Service Registry Numbers (CAS
RNs): 82003-75-6 for the free base, and 79279-03-1 for thehydrochloride salt. It may be noted that in the widely used
‘‘Designer Drugs Directory’’ [41], the CAS RN 79297-03-1
given for acryloyl-a-methylfentanyl (see Fig. 1) in factdenotes acryloylfentanyl hydrochloride. The International
Chemical Identifier Key (InChI Key) for acryloylfentanyl
is RFQNLMWUIJJEQF-UHFFAOYSA-N.The molecular formula of acryloylfentanyl is
C22H26N2O; the monoisotopic mass is 334.2045. Acry-
loylfentanyl contains one basic piperidine-nitrogen atomand thus readily forms salts with organic or inorganic acids.
The reported melting point of the free base is 101–103 "C[42], while the reported melting points of its whitehydrochloride salt are 259–260 "C [36], 252–258 "C (with
decomposition) [37] and 191–194 "C [42, 43].
There are no solubility data on acryloylfentanyl or itssalts; due to its close similarity to fentanyl, the free base is
expected to be sparingly soluble in water; the hydrochlo-
ride and citrate salt are expected to have improved aqueoussolubility. Relevant data for fentanyl are as follows: at
ambient temperature, fentanyl base is poorly soluble in
water (0.032 mg per mL at pH 5.9) [44]; the solubility of
the citrate salt of fentanyl in water is*25 mg/mL, while in
ethanol it is 7.1 mg/mL [45].Like fentanyl, acryloylfentanyl is highly lipophilic, as
indicated by their comparable calculated 1-octanol/water
partition coefficients (cLogP). The respective cLogP valuesfor acryloylfentanyl and fentanyl are 4.13 and 3.89, as
calculated by the ACD/ChemSketch 2015 release version
(Advanced Chemistry Development Inc., Toronto,Canada). The respective cLogP values as calculated by
StarDrop version 6.3.1 software (Optibrium Ltd, Cam-bridge, UK) for acryloylfentanyl and fentanyl are 3.61 and
3.89. The measured LogP value for fentanyl is 4.05 [46].
Due to its high lipophilicity, cross-contamination withtraces of the substance during sample handling and analysis
can be problematic [47].
Synthesis of acryloylfentanyl
There are two published syntheses of acryloylfentanyl,
which involve the acylation of the common precursor
4-anilino-N-phenethylpiperidine (4-ANPP) with acryloylchloride [36, 37] or 3-chloropropionyl chloride [43]
(Fig. 2). The actual synthetic route used for the manufac-
ture of acryloylfentanyl detected on the drug market isunknown, but it most likely relies on precursors and syn-
thetic methods similar to the routes mentioned above or to
those used for the manufacture of pharmaceutical fentanyl[48–52]. A powder sample in a capsule seized in Denmark
was shown to consist of mainly acryloylfentanyl and a
substantial amount of triethylamine hydrochloride (27%)and trace amounts of 4-ANPP, indicating the use of either
of the routes shown in Fig. 2 for the manufacture of the
drug [53].It should be considered that, at least in theory, acry-
loylfentanyl may serve as a precursor to fentanyl by satu-
rating the double bond of the acrylamide moiety usingcatalytic hydrogenation (see, for example, [54]).
Analysis of physical and biological samples
Acryloylfentanyl has been fully characterised by 1H and13C nuclear magnetic resonance spectroscopy
[37, 42, 53, 55], Fourier transform infrared spectroscopy
(both the free base and the HCl salt) [53, 55] and gaschromatography coupled with mass spectrometry
[43, 53, 55]. Quadrupole time-of-flight (QTOF) and
matrix-assisted laser desorption ionization Orbitrap massspectrometric analyses of acryloylfentanyl have also been
described [53]. The ultraviolet and visible spectrum of
acryloylfentanyl have not been reported. A highly sensitive
Forensic Toxicol
123
capillary electrophoresis-electrospray-tandem mass spec-trometry method recently developed for the trace level
analysis of fentanils [56] could be applicable for
acryloylfentanyl.Similar to fentanyl, acryloylfentanyl is not expected to
give a positive response to immunoassay tests developed
for morphine-type opioids. An enzyme-linkedimmunosorbent assay kit developed for fentanyl displayed
strong cross-reactivity (215%) for acryloylfentanyl [57].Acryloylfentanyl did not show cross-reactivity with the
immunoassay panel ABC-multi-10 (Simoco Diagnostic,
Hillerød, Denmark), which includes MDMA [53]. It is notknown whether acryloylfentanyl can be detected by any
presumptive colour tests.
Recently, a liquid chromatography (LC) screening incombination with a high-resolution tandem mass spec-
trometry method was developed for the qualitative and
quantitative forensic analysis of acryloylfentanyl and otherfentanils and their human metabolites with a detection limit
of\5 ng/mL [58]. A method employing LC–QTOF for the
identification of in vitro and in vivo metabolites of acry-loylfentanyl and three other fentanils has also been
described [59].
Pharmacology: mode of action and metabolism
Antinociceptive activity and toxic effectsin the mouse
There have been two studies investigating the antinoci-
ceptive activity of acryloylfentanyl in the mouse
[36, 42, 43]. The first publication mentioning acry-loylfentanyl describes an extensive structure-activity rela-
tionship study involving 22 fentanyl analogues, with
morphine and fentanyl as comparative standards [36]. The
antinociceptive activities of morphine, fentanyl and acry-loylfentanyl in mice upon intraperitoneal (ip) administra-
tion are shown in Table 1. As Table 1 indicates, in this
rodent model of analgesia, acryloylfentanyl is about 170times more effective as an antinociceptive agent than
morphine, though somewhat less potent than fentanyl.
Essawi [42, 43] studied five fentanyl analogues,including acryloylfentanyl, as potential receptor affinity
labels and antinociceptive agents in the mouse using thehot-plate assay; morphine and fentanyl were the compar-
ative standards. Upon ip administration at doses below
1 mg/kg, acryloylfentanyl was a more potent antinocicep-tive agent than fentanyl: while the effect of fentanyl at
doses of 0.1, 0.2 and 0.5 mg/kg dropped considerably at
60–70 min and became insignificant at 90–100 min aftertreatment, ‘‘at comparable doses, acryloylfentanyl main-
tained considerable analgesia at 90 and 120 min after
administration. In its duration, the time-response profile ofacryloylfentanyl resembled more closely that of morphine
(20 mg/kg) than that of fentanyl’’. Remarkably, at doses of
6.8 and 17 mg/kg, the antinociceptive effect of acry-loylfentanyl was sustained up to 4.5 h without signs of
opioid toxicity. At the 25-mg/kg dose, motor activity was
Fig. 2 Synthesis ofacryloylfentanyl fromN-phenyl-1-(2-phenylethyl)-piperidin-4-amine (4-ANPP)and acryloyl chloride [36](top) or 3-chloropropionylchloride [43] (bottom; theintermediate3-chloropropionamidederivative shown in brackets isnot isolated)
Table 1 Antinociceptive activities of morphine, fentanyl and acry-loylfentanyl in mice upon intraperitoneal administration [36]
Compound ED50 (mg/kg) Potency ratio to morphine
Morphine 13.9 1
Fentanyl 0.062 224
Acryloylfentanyl 0.082 169.5
The antinociceptive activity is characterised by the median effectivedose (ED50) calculated by measuring the prolongation of latencytimes of the response to pain (hot-plate test, 55 "C) after adminis-tration of the test substance at various doses as compared to untreatedcontrol
Forensic Toxicol
123
transiently inhibited for 3.5 h. However, at a dose of
50 mg/kg, convulsions developed after 1 h, and ‘‘60%lethality was observed from apparent respiratory depres-
sion’’. Subcutaneous (sc) pre-administration by 30 min of
2 mg/kg naloxone blocked the antinociceptive effect of0.85 mg/kg acryloylfentanyl for about 40 min when the
antagonist effect disappeared, and analgesia and other
morphine-like effects could be noted for about 50 min. Asimilar transient antagonist effect was observed when
naloxone (2 mg/kg, sc) was administered 40 min afteracryloylfentanyl-treatment (0.85 mg/kg, ip): the reversal of
the antinociceptive effect lasted for 70 min, and then
antinociception returned to the same level as beforenaloxone administration. It was concluded that acry-
loylfentanyl ‘‘has a mode of interaction with l-receptorsdifferent from morphine’’.
There is limited information on the acute toxicity of
acryloylfentanyl. In the mouse, ip injection of 25 mg/kg of
the drug caused a transient suppression of motor activity;however, as mentioned above, a dose of 50 mg/kg pro-
duced convulsions 1 h after drug administration, and ‘‘60%
lethality was observed from apparent respiratory depres-sion’’ [43]. Based on this study, the ip LD50 value, that is
the dose required to kill half of the experimental animals,
of acryloylfentanyl may be estimated as between 25 and50 mg/kg. No comparative standard was used in this par-
ticular study. Mouse ip LD50 values reported for fentanyl
were 17.5 [60], 26.3 [61] and 76 mg/kg [62]; for morphine,mouse ip LD50 values of 140 [63] and 340 mg/kg [64] have
been reported. These data indicate that the acute toxicity of
acryloylfentanyl in the mouse is similar to that of fentanyland is higher than that of morphine. There is no informa-
tion on the chronic toxicity of acryloylfentanyl.
Interaction with opioid receptors
Maryanoff et al. [37] determined the binding affinities of aseries of compounds, including acryloylfentanyl, using a
rat brain receptor preparation and tritiated naloxone or
naltrexone as competing receptor ligands (Table 2). Thefentanyl analogues were designed as potential covalent
receptor affinity labels. Morphine, fentanyl and the highly
potent fentanyl analogue (?)-3-methylfentanyl were thecomparative standards. The affinity of the test compounds
to l-opioid receptors was characterised by the half maxi-
mal inhibitory concentration (IC50), that is the molar con-centration of the drug displacing 50% of l-opioid receptor-
preferring tritiated naloxone or naltrexone from the
receptor preparation.As seen in Table 2, the IC50 values obtained for fentanyl
and acryloylfentanyl are practically identical; morphine is
somewhat less effective in inhibiting the binding of radi-olabelled receptor antagonists. The results of this study
indicate that the opioid receptor affinity of acryloylfentanyl
is similar to that of fentanyl and somewhat higher than thatof morphine in this particular rat brain preparation.
Laboratory experiments failed to find evidence for
irreversible binding of acryloylfentanyl to opioid receptors:after incubation, acryloylfentanyl could be completely
‘‘washed out’’ from the receptor preparation; the recovered
receptor was able to bind radiolabelled naltrexone, whichindicates reversible binding of the fentanyl analogue.
Similar non-irreversible binding was observed for anacrylamide derivative of naltrexamine [65]. Thus, the
sustained antinociceptive activity observed by Essawi
[42, 43] is unlikely to be related to covalent binding ofacryloylfentanyl to opioid receptors.
A recent study also confirmed the low reactivity of
compounds containing the acrylamide ‘‘warhead’’, whichhas often been considered as a promiscuous electrophilic
moiety capable of binding to cysteine or other nucleophilic
moieties present in target/off-target receptors and enzymes/other proteins [66]. Nevertheless, there has been an interest
in medicinal chemistry research to develop irreversible
inhibitors containing an acrylamide moiety or relatedelectrophiles [67]; in fact, osimertinib, an N-phenylacry-
lamide derivative-type tyrosine kinase inhibitor, has
received marketing authorisation in the USA and the EU.Because polymerisation processes involving acrylamide
or other acrylic type substances require photochemical or
free radical initiation, spontaneous polymerization ofacryloylfentanyl in the body can be ruled out. Whether any
‘‘biochemical’’ initiator could be involved in the observed
long-lasting activity of acryloylfentanyl in vivo can only bespeculated (see section on animal studies above). No sta-
bility studies have been carried out with acryloylfentanyl.
Other biological activity studies
A search in the PubChem Substance database for biologicalactivity of acryloylfentanyl found 28 test results deposited,
Table 2 Opioid receptor binding data for morphine, fentanyl, acry-loylfentanyl and (?)-3-methylfentanyl [37]
Compound IC50 (nM)
[3H]Naloxone [3H]Naltrexone
Morphine 4.2 27
Fentanyl 1.6 25
Acryloylfentanyl 1.4 17
(?)-3-Methylfentanyl 0.6 1.3
The receptor affinity is expressed by IC50 values, representing theconcentration required for displacement of 50% of tritiated naloxoneor naltrexone as radioligands in a competition assay using rat brainhomogenates
Forensic Toxicol
123
but the substance was found inactive in all assays that
included a range of non-opioid-related targets [68].
Pharmacokinetics
Due to its lipophilicity, acryloylfentanyl, like fentanyl, is
expected to readily cross the blood-brain barrier and also to
diffuse into fat and other tissues (acryloylfentanyl isexpected to have a large volume of distribution).
The pharmacokinetics and the metabolic pathway ofacryloylfentanyl are expected to be similar to those of
fentanyl [69–72] or acetylfentanyl [73–75]. A recent study
determined the structures and relative abundance of themajor acryloylfentanyl metabolites produced by human
hepatocytes in vitro and of those detected in the urine in
five cases of death related to acryloylfentanyl [59]. UsingLC–QTOF analyses, these studies identified altogether 14
biotransformation products, including phase I and II
metabolites. The major metabolites of acryloylfentanyldetected in human urine after hydrolysis of glucuronidated
and/or sulfated phase II conjugates are depicted in Fig. 3.
Similar to the well-studied metabolism of fentanyl[70, 76, 77], the biotransformation involves an oxidative N-
dealkylation, presumably catalysed by cytochrome P450
(CYP450) enzymes, leading to the biologically inactivedesphenethyl metabolite, that is ‘‘nor-acryloylfentanyl’’
(metabolite B1 in the original study). Additional oxidative
metabolic processes were monohydroxylations either onthe alkyl chain of the phenylethyl moiety or on the piper-
idine ring, affording metabolites B9 and B13 (hydroxyla-
tion site not assigned). Dihydroxylation of the aromaticring of the phenylethyl moiety followed by O-
monomethylation of the resulting catechol afforded
metabolite B11 (and, to some extent, its regioisomer; notshown in Fig. 3). Monophenols (B10) and diols, such as
diphenol derivatives of the aniline moiety or a species
dihydroxylated at the acryl moiety (not shown in Fig. 3),
were also detected. Similar to the metabolism of fentanyl,amide hydrolysis (deacylation) affording 4-ANPP (B14)
was a minor pathway. Terminal monohydroxylation of the
acyl chain, producing what is often called ‘‘hydroxyfen-tanyl’’, is a biotransformation step for fentanyl in humans,
but such hydroxylation cannot take place in the case of
acryloylfentanyl. Intact acryloylfentanyl was also presentin the urine of the decedents. The urinary concentrations of
the metabolites were not quantified; the blood acry-loylfentanyl concentration for these five deaths ranged
from 0.05 to 1.20 ng/g.
There is some information on the biological activity oftwo potential metabolites of acryloylfentanyl. An early
study [78] assessing the opioid-like activity of several
fentanyl metabolites in the guinea pig ileum assay foundthat 4-ANPP and 4-anilinopiperidine were less potent than
either fentanyl or morphine by several orders of magnitude.
The only metabolite showing significant activity in thisstudy was a phenolic derivative hydroxylated at the 4-po-
sition of the phenylethyl moiety of fentanyl (for number-
ing, see Fig. 1), the activity of which was found to liebetween morphine and pethidine. Therefore, the corre-
sponding phenolic metabolite of acryloylfentanyl (not
detected in the recent study [59]), if formed, may havesome level of opioid activity and thus may contribute to the
biological, including toxicological, properties of the parent
substance.
Interindividual genetic variability in metabolisingenzymes
For fentanyl, oxidative dealkylation by hepatic CYP450
3A4 and CYP450 3A5 isoenzymes has been demonstrated[71, 76, 79]. The wide variation in the expression of the
genes coding for these CYP450 3A isoenzymes among
Fig. 3 Major humanmetabolites of acryloylfentanyl,with metabolite numberingaccording to originalpublication [59]. Note that dueto incomplete acylation duringmanufacture or to productmislabelling, 4-ANPP couldalso be present in the consumedproducts; thus its detection inbiological matrices might notalways be indicative ofmetabolism
Forensic Toxicol
123
populations is of clinical significance, but the toxicological
consequence of such polymorphisms has not been inves-tigated in fentanyl-overdose cases.
Interactions with other substances, medicines,and other forms of interactions
Should acryloylfentanyl undergo oxidative dealkylation byCYP450 3A4 and CYP450 3A5 isoenzymes, then the use
of this substance with inhibitors of these isoenzymes, suchas clarithromycin, indinavir, ritonavir, saquinavir, itra-
conazole, ketoconazole, nefazodone (all strong CYP3A
inhibitors), erythromycin, fluconazole, grapefruit juice, andverapamil (all moderate CYP3A inhibitors) [80, 81], may
result in prolonged high plasma concentration of acry-
loylfentanyl which could be toxicologically significant, forexample by increasing the risk of potentially fatal respi-
ratory depression.
The concomitant use of other central nervous system(CNS) depressants with opioid analgesics, including other
opioids, sedatives/hypnotics (such as the benzodiazepines
and the z-drugs), ethanol, gabapentinoids (pregabalin andgabapentin), tranquillisers, sedating anti-histamines, and
skeletal muscle relaxants may produce additive depressant
effects [80].Similar to fentanyl, the use of partial opioid agonists/
antagonists (such as buprenorphine, nalbuphine, penta-
zocine) which have high affinity to opioid receptors butrelatively low intrinsic activity could partially antagonise
the effects of acryloylfentanyl and may induce withdrawal
symptoms in people who are opioid dependant [80]; it isunknown if such effects are possibly protective in indi-
viduals poisoned with acryloylfentanyl or other fentanils.
While data are lacking for acryloylfentanyl, the use offentanyl with serotoninergic agents, such as selective
serotonin reuptake inhibitors (the most commonly pre-
scribed antidepressants) or serotonin-norepinephrine reup-take inhibitors or monoamine oxidase inhibitors has been
associated with serotonin syndrome, which is a potentially
life-threatening condition [80, 82]. This association islikely to extend to exposure to illicit drugs which act on the
serotonergic system, such as MDMA and amphetamines.
Unpredictable potentiation by monoamine oxidase inhibi-tors has been reported with some opioid analgesics
[80, 82].
Human data
Routes of administration, dose regimens, and effects
Information on the routes of administration and dose reg-imens is limited, and originated from customs, police
seizures, serious adverse event reports and Internet drug
forums. Acryloylfentanyl may be taken orally as powder incapsules or as tablets; intravenous injection has also been
reported. A novel and apparently popular route of intra-
nasal administration is the use of a metered nasal spray,which delivers approximately 0.1 mL upon one actuation.
Such ready-to-use nasal sprays were sold by online vendors
in Sweden in 2016, and the amount of acryloylfentanyl inthese sprays is typically claimed to be 20 mg as a solution
in a 10-mL spray bottle capable of delivering *100actuations (i.e., *0.2 mg per spray) [58]. In addition, user
reports mention snorting or inhalation by smoking the free
base of acryloylfentanyl; vaping of the drug from an e-ci-garette containing a homemade flavoured e-liquid has also
been mentioned [83]. Injecting users have used marketed
nasal spray solutions or have themselves prepared a solu-tion of the citrate salt of the free base of the drug, some-
times using alcohol as co-solvent. Preparation of a
homemade transdermal patch has also been described [84].From the available limited data, it is not possible to
discern the ‘‘typical’’ dose or dose regimens administered
by users. While a range of doses and regimens have beenreported, these appear to differ depending on factors such
as the route of administration, the user tolerance, the use of
other drugs, and the desired effects. Given that the actualcomposition of the substance sold on the drug market may
differ over time and among geographical areas, the dose
mentioned in such reports is problematic, because thepurity and/or composition of the substance ingested is
typically not known by the user. In fact, it has recently been
reported that some ‘‘akrylfentanyl’’ products sold in Swe-den contained fentanyl instead of acryloylfentanyl [58]. A
confusing factor regarding the dose used is the (probably)
indiscriminate use of ‘‘mg’’ (milligram) and ‘‘lg’’ (mi-crogram) weight units in Internet forums. The difference in
dose by several orders of magnitude could result in a fatal
overdose for an ‘‘uncut’’ product, if such false informationis propagated on the Internet. Given the difficulties in
collecting such data accurately, the information below
should be used with caution.Though human clinical studies are lacking, based on
non-clinical data (see above), it may be assumed that the
opioid-like effects of acryloylfentanyl manifest at dosessimilar to those observed for fentanyl. Those posting self-
reports of use on Internet forums have mentioned that sub-
milligram doses administered by nasal spray were psy-choactive; doses of 0.0027–0.2 mg have been reported. In
comparison, in human clinical trials, acute fentanyl doses
of 0.2 mg intravenously [85], 0.015 mg/kg oral-transmu-cosally (lozenge) [86] or up to 1.6 mg intramuscularly [87]
have been used.
Information on the psychological and behaviouraleffects of acryloylfentanyl is limited to serious adverse
Forensic Toxicol
123
events reported to the EMCDDA [33] and self-reported
experiences from Internet forums. The psychoactivity ofacryloylfentanyl is reportedly similar to that of other opi-
oids and includes relaxation and euphoria. Internet forums
rarely mention side or adverse effects typical of opioids,though some discussions on these sites describe acry-
loylfentanyl as ‘‘longer lasting’’ after dosing than other
new synthetic opioids.
Human poisoning cases
It is important to note that the number of serious adverse
events, including deaths, involving acryloylfentanyl islikely to be underestimated, since testing for this specific
substance is not commonly performed in opioid overdose
cases.
Non-fatal intoxications
So far, Sweden has been the only country that has reported
acryloylfentanyl-related acute intoxications. These cases
relate to presentations to hospital emergency departmentsmade between March and October 2016 [33, 58]. With one
exception, the patients were men, ranging in age from 23 to
51 years; the age of the female patient was 19 years.Acryloylfentanyl was typically administered as nasal spray.
There were eight cases in which acryloylfentanyl was
analytically confirmed to be the sole opioid, with serumand urine acryloylfentanyl concentrations ranging from 0.5
to 2.1 and from 1.8 to 196 ng/mL (creatinine-normalized
concentration: 0.2–10.5 lg/mmol creatinine), respectively.One additional case in Sweden during this period
involved a combination of acryloylfentanyl and 4-chloro-
isobutyrfentanyl that was administered by injection of anextract of pills. Forensic analysis of three other intoxica-
tions that occurred in March 2016 and were believed to
involve acryloylfentanyl identified fentanyl rather thanacryloylfentanyl (see also the section ‘‘Routes of admin-
istration, dose regimens, and effects’’). In some cases,
analysis indicated the intake of alcohol or other licit orillicit substances [33, 58].
The clinical symptoms related to these acute intoxica-
tions appeared to be consistent with the use of an opioidand included decreased consciousness, respiratory depres-
sion and miosis. Other common features were tachycardia
and hypertension. In six of these cases, naloxone wasadministered by paramedics and/or emergency department
personnel [58].
Death cases
A total of 47 analytically confirmed deaths associated withacryloylfentanyl that occurred in 2016 were reported by
three EU member states: Denmark (1 case), Estonia (3),
and Sweden (43) [33]. Detailed information is availableonly for the death cases in Denmark and Sweden. The
decedents were predominantly male (86%), and their age
ranged from 19 to 54 years; the age of the female dece-dents ranged from 29 to 50 years. The deaths typically
occurred in a home environment. Thirty-two of the deaths
occurred between June and August 2016. In at least 40deaths, acryloylfentanyl was either the cause of death or
was likely to have contributed to death (even in the pres-ence of other substances); in two of these deaths, acry-
loylfentanyl was the sole drug present.
Forensic analysis also revealed a range of other sub-stances in the deaths, suggesting that polydrug use was
common. These included a number of CNS depressants
such as benzodiazepines, zopiclone, pregabalin, gaba-pentin, ethanol and cannabinoids (including synthetic
cannabinoids), as well as synthetic cathinones,
amphetamines, antidepressants and antipsychotics. How-ever, acryloylfentanyl was the sole opioid present in 38
cases (86%). In the remaining cases, the opioids detected
were buprenorphine, hydrocodone, oxycodone, 4-fluo-roisobutyrfentanyl and 4-chloroisobutyrfentanyl.
Treatment of poisonings
In suspected acryloylfentanyl overdose cases, naloxone, an
opioid receptor antagonist, should be administered in dosestypical to rescue heroin overdoses [88]. However, given the
short duration of action of naloxone and the high potency
and different pharmacokinetics of acryloylfentanyl, re-peated doses of the antidote may be required to prevent any
reoccurrence of respiratory depression [89, 90]. In six of
the acute acryloylfentanyl intoxication cases that occurredin Sweden (see above), naloxone was administered intra-
venously by paramedics and/or emergency department
personnel at 0.1–0.4 mg, and oxygen supply was provided;one of these cases required continuous infusion for *7 h
during hospital observation; oxygen supply was also doc-
umented in seven of the cases [58].Naloxone has also been used with success to reverse
poisonings caused by other fentanils that have appeared on
the drug market, including 3-methylfentanyl [91],acetylfentanyl [32], furanylfentanyl [30] and butyrylfen-
tanyl [92].
Risks from accidental exposure
It is important to emphasize that accidental exposure, such
as skin contact, inhalation or ingestion, to acryloylfentanyl
and other fentanils poses a serious health risk to the public,law enforcement, medical and forensic laboratory
Forensic Toxicol
123
personnel, postal services and in custodial settings. Where
necessary, such risks should be assessed, and appropriateprocedures, training, and environmental and personal pro-
tective measures should be provided for handling materials
suspected of containing these drugs. This may includetraining in resuscitation and adequate provision of the
opioid antagonist naloxone to reverse accidental poisoning
[27, 89, 93].
Legal status
Acryloylfentanyl is not controlled under the UnitedNations Single Convention on Narcotic Drugs of 1961, as
amended by the 1972 Protocol, or the Convention on
Psychotropic Substances of 1971.In the EU, acryloylfentanyl is controlled under drug
control legislation in Cyprus, Denmark, Estonia, Finland,
Ireland, Latvia, Lithuania, Sweden and the UK; in Austriaand Poland the substance is controlled under specific new
psychoactive substance control legislation. In Turkey, the
substance is also controlled under drug control legislation.In Norway, the importation of, trade in and marketing of
acryloylfentanyl is controlled by the Medicines Act [33].
‘‘Acrylfentanyl’’ has been controlled in China since 1March 2017 [94].
Conclusions
Over the past decade there has been a dramatic worldwideincrease in the number and availability of new psychoactive
substances.Muchof this has beendrivenby the exploitationof
globalization, economic development, and new technologiesby entrepreneurs, where chemical and pharmaceutical com-
panies based in China can produce bulk quantities of new
substances and then cheaply and rapidly ship them to cus-tomers across the world. The fallout from this has been an
increase in reported harms. In this paper we reviewed the case
of acryloylfentanyl, which is one of 20 new fentanils that haveappeared on the drug market in Europe since 2012.
Until recently, acryloylfentanyl was known only from
the scientific literature, but in 2016 this non-controlledsynthetic opioid became available on the illicit drug market
as a powder and nasal spray in Europe and the USA. By the
end of 2016, detection of acryloylfentanyl in six Europeancountries (Denmark, Estonia, Finland, Latvia, Sweden, and
Slovenia1) had been reported to the EMCDDA through the
EU Early Warning System. Chemically, acryloylfentanyl is
an acrylamide derivative of 4-anilinopiperidine and is an
unsaturated analogue of fentanyl, which is a controllednarcotic drug. In non-clinical laboratory studies, it has been
shown to be an opioid receptor agonist and a potent and
long-lasting antinociceptive agent. Typically it is sold as alegal alternative to illicit opioids.
In 2016, acryloylfentanyl was involved in 47 deaths,
with at least 68% of the deaths occurring within a 3-monthperiod. In the majority of the deaths, acryloylfentanyl was
reported to be either the cause of death or to have con-tributed to the death. In addition, more than 20 acute
intoxications suspected to be due to acryloylfentanyl were
reported. The clinical features were generally consistentwith opioid-like toxicity and included life-threatening
effects. Naloxone was shown to be an antidote to poisoning
caused by acryloylfentanyl, though in some cases repeateddoses were required.
One common way of administering acryloylfentanyl
appears to have been with ready-to-use nasal sprays, whichwere sold by online shops. These dosage forms, and others
such as e-liquids, have the potential to make the use of
fentanils easier (as compared to injecting) and moresocially acceptable; these are developments that will
require careful monitoring.
The risks from acryloylfentanyl are not limited to thosewho use the substance. Accidental exposure to the sub-
stance, and in fact to other fentanils, poses a serious risk to
family and friends of users, as well as law enforcement,emergency personnel, medical and forensic laboratory
personnel and those in custodial settings and postal ser-
vices. Specific risks should be identified and appropriaterisk reduction measures implemented. This may include
appropriate protective equipment, training in resuscitation,
and making naloxone readily available to relevant per-sonnel in sufficient quantities in the event of exposure.
As of 1 March 2017, acryloylfentanyl has been con-
trolled under drug control legislation in China. As a result,the open manufacture and sale of this substance may at
least be deterred. Despite this development, it is important
to note that since acryloylfentanyl was first detected inEurope, an additional eight new fentanils have been
detected on the drug market, including 4-fluoroisobutyr-
fentanyl, 4-chloroisobutyrfentanyl, tetrahydrofuranylfen-tanyl and methoxyacetylfentanyl. Based on an analysis of
the literature, there are dozens of other fentanils which
could emerge as new psychoactive substances.In recent years the emergence of hundreds of new psy-
choactive substances on Europe’s drug market has driven
greater complexity into the drug problem. The market ishighly dynamic and demonstrates increasingly innovative
attempts to circumvent regulation. The recent appearance
of a large number of new fentanils as part of this market isof particular concern to public health, given the serious
1 Slovenia reported a sample of light green powder which was a testpurchase from an Internet vendor. The sample was shipped fromChina and was received in May 2016.
Forensic Toxicol
123
acute risk from profound and rapid respiratory depression,
which can lead to apnoea, respiratory arrest and death.In our globalized world, early warning systems should
continue to play a central role in protecting public health
through the early detection and response to such threats.
Acknowledgements The preparation of this publication was sup-ported, in part, by a contract from the European Monitoring Centre forDrugs and Drug Addiction (contract code CT.16.SAT.0099.1.0) toIstvan Ujvary. We thank Ms. Veronika Mikes for interpreting Chinesearticles. The authors would also like to extend their sincere thanks andappreciation to the Early Warning System correspondents of theReitox national focal points and experts from their national earlywarning system networks; the Europol national units and EuropolProject Synergy; Dr. Anders Helander, Department of LaboratoryMedicine and Department of Clinical Pharmacology, KarolinskaInstitutet, Stockholm; Dr. Torben Breindahl, Department of ClinicalBiochemistry, North Denmark Regional Hospital, Aalborg Univer-sity, Hjørring; and Ms. Anabela Almeida, Mr. Andrew Cunninghamand Ms. Paulete Duque at the EMCDDA.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict ofinterest.
Ethical approval For this type of article, formal informed consent isnot required.
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