Metabolism of butyrylfentanyl in fresh human hepatocytes: Chemical synthesis of authentic metabolite standards for definitive identification

Article abstract of DOI:10.1248/bpb.b18-00765

The metabolism of butyrylfentanyl, a new designer drug, was investigated using fresh human hepatocytes isolated from a liver-humanized mouse model. In the culture medium of hepatocytes incubated with butyrylfentanyl, the desphenethylated metabolite (nor-butyrylfentanyl), w-hydroxy-butyrylfentanyl, (w-1)-hydroxy-butyrylfentanyl, 4′-hydroxy-butyrylfentanyl, β-hydroxy-butyrylfentanyl, 4′-hydroxy-3′- methoxy-butyrylfentanyl, and w-carboxy-fentanyl were identified as the metabolites of butyrylfentanyl. Each metabolite was definitively identified by comparing the analytical data with those of authentic standards. The amount of the main metabolite, nor-butyrylfentanyl, reached 37% of the initial amount of butyrylfentanyl at 48 h. W-Hydroxy-butyrylfentanyl and (w-1)-hydroxy-butyrylfentanyl, formed by hydroxylation at the Nbutyryl group of butyrylfentanyl, were the second and third largest metabolites, respectively. The majority of 4′-hydroxy-butyrylfentanyl and 4′-hydroxy-3′-methoxy-butyrylfentanyl was considered to be conjugated. CYP reaction phenotyping for butyrylfentanyl using human liver microsomes and various anti-CYP antibodies revealed that CYP3A4 was involved in the formation of nor-butyrylfentanyl, (w-1)-hydroxybutyrylfentanyl, and β-hydroxy-butyrylfentanyl. In contrast, CYP2D6 was involved in the formation of w-hydroxy-butyrylfentanyl.

Full text of DOI:10.1248/bpb.b18-00765

Vol. 42, No. 4
Biol. Pharm. Bull. 42, 623–630 (2019)
623
Regular Article
Metabolism of Butyrylfentanyl in Fresh Human Hepatocytes: Chemical
Synthesis of Authentic Metabolite Standards for Definitive Identification
Tatsuyuki Kanamori,* Yuko Togawa Iwata, Hiroki Segawa, Tadashi Yamamuro,
Kenji Kuwayama, Kenji Tsujikawa, and Hiroyuki Inoue
National Research Institute of Police Science; 6–3–1 Kashiwanoha, Kashiwa, Chiba 277–0882, Japan.
Received October 1, 2018; accepted January 10, 2019
The metabolism of butyrylfentanyl, a new designer drug, was investigated using fresh human
hepatocytes isolated from a liver-humanized mouse model. In the culture medium of hepatocytes incubated
with butyrylfentanyl, the desphenethylated metabolite (nor-butyrylfentanyl), ω-hydroxy-butyrylfentanyl,
(ω-1)-hydroxy-butyrylfentanyl, 4ꢀ-hydroxy-butyrylfentanyl, β-hydroxy-butyrylfentanyl, 4ꢀ-hydroxy-3ꢀ-
methoxy-butyrylfentanyl, and ω-carboxy-fentanyl were identified as the metabolites of butyrylfentanyl. Each
metabolite was definitively identified by comparing the analytical data with those of authentic standards. The
amount of the main metabolite, nor-butyrylfentanyl, reached 37% of the initial amount of butyrylfentanyl
at 48h. ω-Hydroxy-butyrylfentanyl and (ω-1)-hydroxy-butyrylfentanyl, formed by hydroxylation at the N-
butyryl group of butyrylfentanyl, were the second and third largest metabolites, respectively. The majority
of 4ꢀ-hydroxy-butyrylfentanyl and 4ꢀ-hydroxy-3ꢀ-methoxy-butyrylfentanyl was considered to be conjugated.
CYP reaction phenotyping for butyrylfentanyl using human liver microsomes and various anti-CYP
antibodies revealed that CYP3A4 was involved in the formation of nor-butyrylfentanyl, (ω-1)-hydroxy-
butyrylfentanyl, and β-hydroxy-butyrylfentanyl. In contrast, CYP2D6 was involved in the formation of
ω-hydroxy-butyrylfentanyl.
Key words butyrylfentanyl; metabolism; hepatocyte; CYP reaction phenotyping
butyrylfentanyl using human liver microsomes and recom-
binant CYP, and revealed that butyrylfentanyl underwent
INTRODUCTION
In recent years, the abuse of opioid drugs, especially fen- hydroxylation at the butanamide side chain and aromatic ring
tanyl analogs, has become a serious problem, particularly in and N-dealkylation, and that CYP3A4 and CYP2D6 were in-
North America and Europe, where many opioid-related over- volved in these reactions.5) In addition, they analyzed authen-
dose deaths have been reported.1,2) Butyrylfentanyl is a new tic blood and urine samples, and they concluded that hydrox-
designer drug chemically similar to fentanyl, with an N-butyr- ylation of the butanamide side chain followed by oxidation to
yl group replacing the N-propionyl group of fentanyl (Fig. 1). the carboxylic acid was the major metabolic pathway of butyr-
The pharmacological activity of butyrylfentanyl is reportedly ylfentanyl in vivo. Staeheli et al. analyzed blood, urine, and
13% that of fentanyl.3) In the United States, seven butyrylfen- organ samples from a fatal case of butyrylfentanyl poisoning,
tanyl cases were reported for the first time in 2014, and the and they also reported that carboxy- and hydroxy-butyrylfen-
number of reports increased to 81 in 2015.4) Butyrylfentanyl tanyl were the most abundant metabolites of butyrylfentanyl.6)
is used as a substitute for heroin or prescription opioids; how- However, they did not use authentic standards to identify and
ever, the identity, purity, and quantity of illicit butyrylfentanyl quantitate the metabolites of butyrylfentanyl; therefore, defini-
is uncertain and inconsistent, posing serious adverse health tive identification and accurate quantitation of the metabolites
risks to abusers. To avoid the imminent risks caused by butyr- of butyrylfentanyl has not been accomplished yet.
ylfentanyl, the United States Drug Enforcement Administra-
tion designated this drug as a schedule I substance under the ylfentanyl using fresh human hepatocytes isolated from a
Controlled Substances Act in 2016.4) liver-humanized mouse model (PXB-cells)7) and synthesized
In this study, we investigated the metabolism of butyr-
Several studies on the metabolism of butyrylfentanyl have the putative metabolites chemically to identify the metabo-
been published. Steuer et al. investigated the metabolism of lites. In addition, we performed CYP reaction phenotyping
using human liver microsomes and anti-CYP antibodies to
clarify which CYP isoform is involved in the metabolism of
butyrylfentanyl.
MATERIALS AND METHODS
Chemicals and Reagents Authentic standards of bu-
tyrylfentanyl, thiofentanyl, and the metabolites of butyrylfenta-
nyl were synthesized in our laboratory. PXB-cells (seeded in a
24-well plate, 2.1×105 cells/cm2) and the culture medium for
PXB-cells were purchased from PhoenixBio (Higashihiro-
Fig. 1. Chemical Structures of Butyrylfentanyl and Fentanyl
*To whom correspondence should be addressed. e-mail: kanamori@nrips.go.jp
© 2019 The Pharmaceutical Society of Japan

624
Biol. Pharm. Bull.
Vol. 42, No. 4 (2019)
shima, Japan); Helix pomatia β-glucuronidase/aryl sulfatase
1H-NMR (CDCl3) δ: 0.80 (3H, t, J=7.2Hz), 1.25 (2H, qd,
(β-glucuronidase, 32units/mL; aryl sulfatase, 102units/mL) J=4.1, 12.4Hz), 1.52–1.61 (2H, m), 1.78 (2H, d, J=11.4Hz),
was purchased from Merck (Darmstadt, Germany); anti-CYP 1.88 (2H, t, J=7.5Hz), 2.72 (2H, td, J=2.2, 12.2Hz),
antibodies (anti-CYP1A2, CYP2C8, CYP2C9, CYP2C19, 3.02–3.07 (2H, m), 4.71–4.78 (1H, m).
CYP2D6, and CYP3A4) and preimmune rabbit immunoglobu-
lin G (IgG) were purchased from CYP450-GP (Vista, CA,
U.S.A.); and human liver microsomes (Ultrapool HLM 150,
Nor-butyrylfentanyl Hydrochloride
1H-NMR (CD3OD) δ: 7.22–7.26 (2H, m), 7.47–7.54 (3H, m).
N-Phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]-4-hydroxy-
6.6nmol P450/mL) were purchased from Corning (Corning, butanamide (ω-Hydroxy-butyrylfentanyl) Despropionyl-
NY, U.S.A.). All other reagents used were of analytical grade. fentanyl (100mg), 4-benzyloxybutyric acid (90mg), HATU
Synthesis of Authentic Standards of Butyrylfentanyl and (180mg), and triethylamine (100µL) in acetonitrile (6mL)
Its Metabolites All synthesized standards were confirmed were refluxed for 7h. The solvent was evaporated under
by positive electrospray ionization (ESI) mass spectrom- vacuum and the residue was purified by flash chromatogra-
1
etry and H-NMR. ESI MS were obtained from an ion trap phy (column, silica gel 12g; solvent, chloroform–methanol).
mass spectrometer (LCQ FLEET, Thermo Fisher Scientific, The purified product was dissolved in methanol, acidified
1
Waltham, MA, U.S.A.). H-NMR spectra were measured on with concentrated hydrochloric acid, and then stirred with
an NMR spectrometer (JNM-ECA600, JEOL, Akishima, palladium-activated carbon (20mg, Pd 5%) under a hydrogen
Japan) using tetramethylsilane as an internal standard (IS).
atmosphere for 7d. Water was added to the reaction mixture
N-Phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]butanamide and the solution was basified with 28% ammonium hydroxide
(Butyrylfentanyl) Butyrylfentanyl was synthesized accord- solution then extracted with chloroform–2-propanol (3:1).
ing to the method of Siegfried.8) Briefly, 1-(2-phenylethyl)- The solvent was evaporated to dryness under vacuum and the
4-piperidone was condensed with aniline in the presence of residue was purified by flash chromatography (column, silica
4A molecular sieves. The product was reduced using sodium gel 4g; solvent, chloroform–methanol). The amine product
borohydride to obtain N-phenyl-1-(2-phenylethyl)-4-piperidin- was converted to the hydrochloride salt as described above
amine (despropionylfentanyl). Despropionylfentanyl was bu- to obtain N-phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]-4-hy-
tyrylated using butyric anhydride to give butyrylfentanyl. The droxybutanamide (ω-hydroxy-butyrylfentanyl) hydrochloride
butyrylfentanyl free base was converted to the hydrochloride (11.2mg) as a white solid.
salt by the addition of concentrated hydrochloric acid–metha-
nol (1:9), then the hydrochloride salt was precipitated by the
ω-Hydroxy-butyrylfentanyl (Free Base)
1H-NMR (CDCl3) δ: 1.44 (2H, qd, J=3.5, 12.2Hz),
addition of diethylether to obtain butyrylfentanyl hydrochlo- 1.75–1.84 (4H, m), 2.10 (2H, t, J=6.6Hz), 2.16 (2H, t,
ride as a white powder.
J=11.1Hz), 2.51–2.57 (2H, m), 2.70–2.75 (2H, m), 3.00 (2H,
d, J=11.4Hz), 3.58–3.63 (2H, m), 4.63–4.71 (1H, m).
ω-Hydroxy-butyrylfentanyl Hydrochloride
Butyrylfentanyl (Free Base)
1H-NMR (CDCl3) δ: 0.80 (3H, t, J=7.2Hz), 1.43 (2H, qd,
J=3.0, 12.0Hz), 1.52–1.61 (2H, m), 1.81 (2H, d, J=10.8 Hz),
1H-NMR (CD3OD) δ: 7.23–7.29 (5H, m), 7.30–7.34 (2H, m),
1.89 (2H, t, J=7.5Hz), 2.16 (2H, t, J=11.4Hz), 2.50–2.58 7.47–7.55 (3H, m).
(2H, m), 2.70–2.77 (2H, m), 3.00 (2H, d, J=11.4Hz),
4.66–4.73 (1H, m).
N-Phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]-3-hydroxy-
butanamide ((ω-1)-Hydroxy-butyrylfentanyl) Despropion-
Butyrylfentanyl Hydrochloride
ylfentanyl (100mg), 3-butenoic acid (72mg), HATU (180mg),
1H-NMR (CD3OD) δ: 7.23–7.27 (5H, m), 7.30–7.34 (2H, m), and triethylamine (100µL) in acetonitrile (6mL) were refluxed
7.48–7.55 (3H, m).
for 20h. The solvent was evaporated under vacuum, and then
N-Phenyl-N-(4-piperidinyl)butanamide (Nor-butyrylfen- the residue was purified by flash chromatography (column,
tanyl) 1-Benzyl-4-piperidone was condensed with aniline silica gel 12g; solvent, chloroform–ethyl acetate). The purified
and was reduced using sodium borohydride as described product (44mg) dissolved in tetrahydrofuran (1mL) was added
above to give N-phenyl-1-benzylpiperidin-4-amine. N- to a solution of mercury(II) acetate (120mg) in 10% acetic
Phenyl-1-benzylpiperidin-4-amine (0.319g), butyric anhydride acid (1mL), and the solution was heated at 90°C for 20h.
(1.25g), and triethylamine (0.34g) in ethyl acetate (10mL) After the reaction was complete, 0.5M sodium borohydride
were refluxed for 21h. The solvent was removed under vac- (0.4mL) in 3M sodium hydroxide solution was added, and
uum, water was added to the residue, and the solution was then sodium chloride was added until the solution separated
basified with sodium hydroxide solution then extracted with into two layers. The tetrahydrofuran layer was transferred to a
chloroform. The extract was evaporated to dryness under separating funnel, water was added, and the solution was basi-
vacuum and the residue was purified by flash chromatogra- fied with 28% ammonium hydroxide solution and extracted
phy (column, silica gel 24g; solvent, n-hexane–ethyl acetate) with chloroform. The solvent was evaporated to dryness
to give N-phenyl-N-(1-benzyl-4-piperidinyl)butanamide. The under vacuum, and then the residue was purified by prepara-
purified N-phenyl-N-(1-benzyl-4-piperidinyl)butanamide was tive TLC (plate, silica gel; solvent, ethyl acetate). The amine
dissolved in methanol (5mL), and the solution was acidified product was converted to the hydrochloride salt as described
with concentrated hydrochloric acid. Palladium-activated car- above to obtain N-phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]-
bon (60mg, Pd 5%) was added to the solution and the mixture 3-hydroxybutanamide ((ω-1)-hydroxy-butyrylfentanyl) hydro-
was stirred for 5d under a hydrogen atmosphere. The solution chloride (14.3mg) as a white solid.
was filtered and evaporated to dryness under vacuum, dried
in a desiccator to give N-phenyl-N-(4-piperidinyl)butanamide
(nor-butyrylfentanyl) hydrochloride (0.292g) as a white solid.
Nor-butyrylfentanyl (Free Base)
(ω-1)-Hydroxy-butyrylfentanyl (Free Base)
1H-NMR (CDCl3) δ: 1.04 (3H, d, J=6.6Hz), 1.40–1.49 (2H,
m), 1.78–1.84 (2H, m), 1.96 (1H, dd, J=9.6, 16.8Hz), 2.05 (1H,
dd, J=2.7, 16.5Hz), 2.16 (2H, t, J=12.0Hz), 2.51–2.56 (2H,

Vol. 42, No. 4 (2019)
Biol. Pharm. Bull.
625
m), 2.70–2.75 (2H, m), 3.01 (2H, d, J=11.4Hz), 4.06–4.13 4-piperidinyl]butanamide (4′-Hydroxy-3′-methoxy-butyryl-
(1H, m), 4.63–4.71 (1H, m).
fentanyl) A sample of 4′-hydroxy-3′-methoxy-butyrylfentanyl
(ω-1)-Hydroxy-butyrylfentanyl Hydrochloride
hydrochloride (9.8mg) was prepared from nor-butyrylfentanyl
1H-NMR (CD3OD) δ: 7.23–7.29 (5H, m), 7.30–7.34 (2H, m), hydrochloride (40mg) by the method used for the synthesis of
7.48–7.55 (3H, m).
4′-hydroxy-butyrylfentanyl.
N-Phenyl-N-[1-(2-hydroxy-2-phenylethyl)-4-piperidinyl]-
butanamide (β-Hydroxy-butyrylfentanyl) A mixture of
4′-Hydroxy-3′-methoxy-butyrylfentanyl (Free Base)
1H-NMR (CDCl3) δ: 0.80 (3H, t, J=7.2Hz), 1.38–1.47
nor-butyrylfentanyl hydrochloride (40mg), phenacyl chloride (2H, m), 1.52–1.61 (2H, m), 1.80 (2H, d, J=12.0Hz), 1.89
(30mg), and sodium bicarbonate (16mg) in water–2-butanone (2H, t, J=7.5Hz), 2.15 (2H, t, J=11.4Hz), 2.47–2.53 (2H,
(1.2mL, 1:5) was heated at 90°C for 3h. Water was added to m), 2.62–2.69 (2H, m), 2.98 (2H, d, J=11.4Hz), 3.83 (3H, s),
the reaction mixture, and the solution was basified with 28% 4.66–4.73 (1H, m).
ammonium hydroxide solution then extracted with ethyl ace-
tate. The solvent was evaporated to dryness under vacuum and
4′-Hydroxy-3′-methoxy-butyrylfentanyl Hydrochloride
1H-NMR (CD3OD) δ: 6.67 (1H, dd, J=2.4, 8.4Hz), 6.73
the residue was dissolved in methanol (3mL). Sodium borohy- (1H, d, J=7.2Hz), 6.81 (1H, d, J=2.4Hz), 7.25 (2H, d,
dride (30mg) was added to the solution and it was stirred for J=7.2Hz), 7.47–7.55 (3H, m).
10min. Water was added to the reaction mixture, the solution
was basified with 28% ammonium hydroxide solution, and propanamide (ω-Carboxy-fentanyl)
N-Phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]-3-carboxy-
mixture of de-
A
then extracted with chloroform. The solvent was evaporated to spropionylfentanyl (50mg), methyl 4-chloro-4-oxobutyrate
dryness under vacuum and the residue was purified by prepar- (40mg), and triethylamine (30µL) in dichloromethane (2mL)
ative TLC (plate, silica gel; solvent, ethyl acetate). The amine was stirred on ice for 1h. Zero point one moler hydrochloric
product was converted to the hydrochloride salt as described acid was added to the reaction mixture, and the mixture was
above to obtain N-phenyl-N-[1-(2-hydroxy-2-phenylethyl)-4- extracted with chloroform. The solvent was evaporated to dry-
piperidinyl]butanamide (β-hydroxy-butyrylfentanyl) hydro- ness under vacuum, and the residue was dissolved in 0.1M
chloride (27.1mg) as a white powder.
hydrochloric acid (2mL) and refluxed for 2h. The reaction
mixture was basified with 10M sodium hydroxide solution,
β-Hydroxy-butyrylfentanyl (Free Base)
1H-NMR (CDCl3) δ: 0.80 (3H, t, J=7.8Hz), 1.33–1.48 and then washed with chloroform. The aqueous layer was
(2H, m), 1.53–1.61 (2H, m), 1.77–1.85 (2H, m), 1.89 (2H, t, acidified with 3M hydrochloric acid then the pH was ad-
J=7.2Hz), 2.18 (1H, t, J=11.1Hz), 2.37 (1H, t, J=11.7Hz), justed to 9 with 28% ammonium hydroxide solution before
2.42–2.49 (2H, m), 2.79 (1H, d, J=10.2Hz), 3.15 (1H, d, extracting with chloroform–2-propanol (3:1). The organic
J=9.6Hz), 4.63 (1H, dd, J=3.0, 10.2Hz), 4.66–4.72 (1H, m).
β-Hydroxy-butyrylfentanyl Hydrochloride
layer was evaporated to dryness under vacuum, and then the
residue was purified by flash chromatography (column, silica
1H-NMR (CD3OD) δ: 7.26 (2H, d, J=7.2 Hz), 7.29–7.33 gel 4g; solvent, chloroform-methanol) to obtain N-phenyl-
(1H, m), 7.35–7.42 (4H, m), 7.48–7.56 (3H, m).
N-[1-(2-phenylethyl)-4-piperidinyl]-3-carboxypropanamide
N-Phenyl-N-[1-[2-(4-hydroxyphenyl)ethyl]-4-piperidinyl]- (ω-carboxy-fentanyl) (39.7mg) as a clear oil.
butanamide (4′-Hydroxy-butyrylfentanyl) A mixture of nor-
butyrylfentanyl hydrochloride (40mg), 2-(4-benzyloxyphenyl)-
ω-Carboxy-fentanyl (Free Base)
1H-NMR (CDCl3) δ: 1.45–1.54 (2H, m), 1.82 (2H, d,
ethyl methanesulfonate (92mg), and potassium carbonate J=10.8Hz), 2.17 (2H, t, J=11.4Hz), 2.26 (2H, t, J=6.0Hz),
(90mg) in 2-butanone (4mL) was heated at 90°C for 20h. 2.52–2.59 (4H, m), 2.70–2.76 (2H, m), 3.03 (2H, d, J=10.2 Hz),
2-(4-Benzyloxyphenyl)ethyl methanesulfonate was prepared 4.60–4.67 (1H, m).
from 2-(4-benzyloxyphenyl)ethanol by a previously reported
ω-Carboxy-fentanyl (Free Base)
method.9) Water was added to the reaction mixture and the
1H-NMR (CD3OD) δ: 7.17–7.21 (3H, m), 7.24–7.30 (4H, m),
mixture was extracted with chloroform. The solvent was 7.44–7.52 (3H, m).
evaporated to dryness under vacuum, and then the residue
Incubation of Drug with PXB-Cells PXB-cells seeded
was purified by flash chromatography (column, silica gel in a 24-well plate were incubated for 11d at 37°C and 5%
4g; solvent, n-hexane–ethyl acetate). The purified product CO2 before addition of the drug. Culture medium was re-
(25.5mg) was dissolved in methanol, acidified with concen- placed every 2d. Butyrylfentanyl hydrochloride (dissolved in
trated hydrochloric acid, and then stirred with palladium- phosphate-buffered saline) was added to the cells at a final
activated carbon (5mg, Pd 5%) under a hydrogen atmosphere concentration of 10µM, and then the cells were continuously
for 3d to obtain N-phenyl-N-[1-[2-(4-hydroxyphenyl)ethyl]-4- incubated. The medium was sampled 24 and 48h after the
piperidinyl]butanamide (4′-hydroxy-butyrylfentanyl) hydro- addition of the drug, and was stored at −20°C until analysis.
chloride (20.1mg) as a white solid.
Identification of the Metabolites A sample of the culture
medium (25µL) was incubated with 0.25M acetate buffer
4′-Hydroxy-butyrylfentanyl (Free Base)
1H-NMR (CDCl3) δ: 0.80 (3H, t, J=7.2Hz), 1.37–1.47 (2H, (15 µL, pH 5.0) containing β-glucuronidase/aryl sulfatase
m), 1.52–1.61 (2H, m), 1.79 (2H, d, J=11.4Hz), 1.89 (2H, (β-glucuronidase, 0.01 unit) at 60°C for 1.5h. Acetonitrile
t, J=7.5Hz), 2.11 (2H, t, J=11.7Hz), 2.45–2.51 (2H, m), (250 µL) was added to the hydrolyzed sample, and the mix-
2.62–2.67 (2H, m), 2.98 (2H, d, J=10.8Hz), 4.64–4.71 (1H, ture was vortexed for 5s, and then centrifuged at 10000×g
m).
4′-Hydroxy-butyrylfentanyl Hydrochloride
for 5min. The supernatant was evaporated to dryness under
a gentle stream of nitrogen, and the residue was dissolved in
1H-NMR (CD3OD) δ: 6.71–6.74 (2H, m), 7.05 (2H, d, the initial mobile phase (50µL) for LC/MS analysis. LC/MS
J=9.0Hz), 7.26 (2H, d, J=7.2Hz), 7.47–7.55 (3H, m).
analyses were carried out using an LC system (Accela, Ther-
N-Phenyl-N-[1-[2-(4-hydroxy-3-methoxyphenyl)ethyl]- mo Fisher Scientific) connected to the LCQ FLEET ion trap

626
Biol. Pharm. Bull.
Vol. 42, No. 4 (2019)
Table 1. Monitoring Ions and Collision Energies for LC/MS* Analysis
of the Compounds
Monitoring ion (m/z)
Collision
Compound
energy
(eV)
Precursor
Product
([M+H]+)
Butyrylfentanyl
351.1
247.1
367.1
367.1
367.1
367.1
188.2
84.2
23
18
23
23
33
21
32
24
33
Nor-butyrylfentanyl
ω-Hydroxy-butyrylfentanyl
(ω-1)-Hydroxy-butyrylfentanyl
4′-Hydroxy-butyrylfentanyl
β-Hydroxy-butyrylfentanyl
188.2
188.2
121.1
204.2
151.2
188.2
111.1
4′-Hydroxy-3′-methoxy-butyrylfentanyl 397.1
ω-Carboxy-fentanyl
381.1
Thiofentanyl (IS)
343.1
Fig. 2. TIC and EICs Obtained from the Medium of the Hepatocytes
Cultured with Butyrylfentanyl for 48h
*Triple quadrupole mass spectrometer was used for quantitative analysis.
1, butyrylfentanyl; 2, nor-butyrylfentanyl; 3, ω-hydroxy-butyrylfentanyl; 4,
(ω-1)-hydroxy-butyrylfentanyl; 5, 4′-hydroxy-butyrylfentanyl; 6, β-hydroxy-
butyrylfentanyl; 7, ω-carboxy-fentanyl; 8, 4′-hydroxy-3′-methoxybutyrylfentanyl.
mass spectrometer. The conditions were as follows: column,
CORTECS C18 (2.1×50mm; particle diameter, 2.7µm; Wa-
ters, Milford, MA, U.S.A.) maintained at 40°C; mobile phase
composition, 0.1% formic acid (A) and methanol (B); linear
gradient mode, 20% B for 1min, 20 to 80% B over 8min,
80% B for 2min, and 80 to 20 B over 0.1min; flow rate,
0.2mL/min; MS interface, positive ESI; analysis mode, scan
(m/z 100–500) and product ion analysis (normalized collision
energy, 35%; precursor ions, protonated molecule of each
compound).
Quantitation of the Metabolites A sample of the culture
medium (25µL) was hydrolyzed as described above. The IS
(10 µL) solution (thiofentanyl hydrochloride, 50ng/10µL water)
and acetonitrile (250µL) were added to the reaction mixture
and the mixture was vortexed for 5s. After centrifugation at
10000×g for 5min, the supernatant (50µL) was mixed with
0.1% formic acid (200µL), and centrifuged at 10000×g for
5min. The supernatant was analyzed by LC/MS using an LC
system (NANOSPACE SI-2, Shiseido, Tokyo, Japan) con-
nected to a triple quadrupole mass spectrometer (TSQ Quan-
tum, Thermo Fisher Scientific). The column, mobile phase
composition, flow rate, and MS interface were the same as for
the identification of the metabolites. The analysis mode was
selected reaction monitoring (SRM). The SRM parameters are
listed in Table 1.
Authentic standards of butyrylfentanyl and its metabolites
were added to the culture medium and processed as described
above, omitting the hydrolysis step, to obtain the calibration
curves. Excellent linearity was obtained over the concentra-
tion range 0.016–10µM for each compound, with a correlation
coefficient of 0.99.
CYP Reaction Phenotyping CYP reaction phenotyp-
ing was performed according to the protocol provided by the
manufacturer. Briefly, 0.1M potassium phosphate buffer (KPi,
pH 7.4), human liver microsomes, and an anti-CYP antibody
were mixed in a test tube and incubated at 37°C for 3min. As
a control, preimmune rabbit IgG was used instead of the anti-
CYP antibody. After standing at room temperature for 10min,
1M KPi, water, butyrylfentanyl hydrochloride solution,
and a reduced nicotinamide adenine dinucleotide phosphate
(NADPH)-generating system (mixture of glucose-6-phos-
Fig. 3. Product Ion Spectra of Authentic Standards of Butyrylfentanyl
and Its Metabolites Obtained by the Ion Trap Mass Spectrometer
1, butyrylfentanyl; 2, nor-butyrylfentanyl; 3, ω-hydroxy-butyrylfentanyl; 4,
(ω-1)-hydroxy-butyrylfentanyl; 5, 4′-hydroxy-butyrylfentanyl; 6, β-hydroxy-
butyrylfentanyl; 7, ω-carboxy-fentanyl; 8, 4′-hydroxy-3′-methoxybutyrylfentanyl.
phate, glucose-6-phosphate dehydrogenase, and NADP+
in
water) were added to the test tube and incubated at 37°C for

Vol. 42, No. 4 (2019)
Biol. Pharm. Bull.
627
30min. The volume of each reaction mixture was 200µL. The proposed metabolic pathways for butyrylfentanyl are shown
final concentrations of the components in the reaction mix- in Fig. 4.
ture were as follows: butyrylfentanyl 50µM, KPi 0.1M, CYP
CYP Reaction Phenotyping To clarify which isoform
0.1 µM, glucose-6-phosphate 10mM, glucose-6-phosphate of CYP was involved in the formation of each metabolite of
dehydrogenase 1U/mL, NADP+ 0.5mM, and anti-CYP anti- butyrylfentanyl, CYP reaction phenotyping was performed.
body 0.075–0.75mg/mL. After incubation, IS solution (10µL; Figure 5 shows the inhibition of the formation of each me-
thiofentanyl hydrochloride, 50ng/10µL water) and acetonitrile tabolite of butyrylfentanyl by various anti-CYP antibodies.
(0.8mL) were added and the mixture was vortexed for 5s. A None of the five metabolites in Fig. 5 were formed without
portion of the supernatant was diluted five times with 0.1% NADPH, indicating that these metabolites were formed by
formic acid and centrifuged at 10000×g for 5min. The su- NADPH-dependent enzyme(s) (data not shown). The forma-
pernatant was analyzed using the LC-triple quadrupole mass tion of nor-butyrylfentanyl and β-hydroxy-butyrylfentanyl
spectrometer as described above to quantitate the metabolites. were markedly inhibited by the anti-CYP3A4 antibody, in-
dicating that CYP3A4 was involved in the majority of the
formation of these metabolites. In contrast, the formation of
(ω-1)-hydroxy-butyrylfentanyl was inhibited by half by the
anti-CYP3A4 antibody, implying that CYP3A4 plus another
NADPH-dependent enzyme (possibly another CYP) produced
this metabolite. In contrast, the formation of ω-hydroxy-
butyrylfentanyl was inhibited by the anti-CYP2D6 antibody,
instead of the anti-CYP3A4 antibody. The production of
4′-hydroxy-butyrylfentanyl was not inhibited by any of the
anti-CYP-antibodies tested. According to our previous study,
4′-hydroxy-fentnayl is formed from fentanyl by CYP2D6.10)
RESULTS AND DISCUSSION
Identification of the Metabolites Because fresh human
hepatocytes isolated from a liver-humanized mouse model
(PXB-cells) have high activities of phase I and II drug-metab-
olizing enzymes, we used these cells in studying the metabo-
lism of butyrylfentanyl. The total ion current chromatogram
(TIC) and extracted ion chromatograms (EICs) obtained from
the medium of hepatocytes cultured with butyrylfentanyl
for 48h are shown in Fig. 2. Unmetabolized butyrylfentanyl
(peak 1) and seven metabolites (peak 2, nor-butyrylfentanyl; A different enzyme may be involved in the 4′-hydroxylation
peak 3, ω-hydroxy-butyrylfentanyl; peak 4, (ω-1)-hydroxy- of butyrylfentanyl. Steuer et al.5) reported that CYP3A4 and
butyrylfentanyl; peak 5, 4′-hydroxy-butyrylfentanyl; peak CYP2D6 are involved in the formation of nor-butyrylfentanyl
6, β-hydroxy-butyrylfentanyl; peak 7, ω-carboxy-fentanyl; and ω-hydroxy-butyrylfentanyl, respectively, and their results
peak 8, 4′-hydroxy-3′-methoxy-butyrylfentanyl) were detected are consistent with our results.
in the EICs. All metabolites were confirmed by comparing
Metabolite Profile of Butyrylfentanyl in PXB-Cells The
their retention times and mass spectra with those of the au- metabolite profile of butyrylfentanyl in PXB-cells is shown
thentic standards. The product ion spectra of the compounds in Fig. 6. After 24 and 48h incubation of butyrylfentanyl
are shown in Fig. 3. Among the hydroxylated metabolites with PXB-cells, 23 and 13% of butyrylfentanyl remained in
(peaks 3–6), the spectra of ω-hydroxy-butyrylfentanyl and the culture medium, respectively. The amount of the main
(ω-1)-hydroxy-butyrylfentanyl (peaks 3, 4) were similar to metabolite of butyrylfentanyl formed by PXB-cells, nor-butyr-
each other, whereas those of 4′-hydroxy-butyrylfentanyl ylfentanyl, reached 37% of the initial amount of butyrylfen-
and β-hydroxy-butyrylfentanyl (peaks 5 and 6) were easily tanyl at 48h. The second and third largest metabolites were
distinguished from the other hydroxylated metabolites. The (ω-1)-hydroxy-butyrylfentanyl and ω-hydroxy-butyrylfentanyl,
Fig. 4. Proposed Metabolic Pathways for Butyrylfentanyl

628
Biol. Pharm. Bull.
Vol. 42, No. 4 (2019)
respectively, and 4′-hydroxy-butyrylfentanyl, β-hydroxy- our in vitro results were not consistent with their in vivo
butyrylfentanyl, ω-carboxy-fentanyl, and 4′-hydroxy-3′- results. ω-Carboxy-fentanyl was formed from ω-hydroxy-
methoxy-butyrylfentanyl were the minor metabolites. The butyrylfentanyl by further oxidation via an aldehyde; the
amount of each metabolite increased with the incubation time candidate enzymes involved in these reactions are alcohol
(24h<48h), except for β-hydroxy-butyrylfentanyl; this metab- dehydrogenase and aldehyde dehydrogenase. The low activity
olite might undergo further metabolism, such as hydroxylation of these enzymes in PXB-cells may explain why only small
and/or N-dealkylation, in PXB-cells.
amounts of ω-carboxy-fentanyl were formed in the PXB-
Steuer et al.5) and Staeheli et al.6) reported that ω-hydroxy- cells. It is unclear what made such difference in the amount
butyrylfentanyl and ω-carboxy-fentanyl are the major me- of nor-butyrylfentanyl between in PXB-cells and in vivo. One
tabolites of butyrylfentanyl in biological specimens, such as possibility is that the activity of CYP2D6 was relatively lower
blood and urine, obtained from butyrylfentanyl users, whereas in PXB-cells than in vivo, resulted in producing less amount
nor-butyrylfentanyl is the minor metabolite. The detected of CYP2D6-mediated metabolite (ω-hydroxy-butyrylfentanyl)
amounts of nor-butyrylfentanyl and ω-carboxy-fentanyl in and more amounts of CYP3A4-mediated metabolites (nor-
Fig. 5. Inhibition of the Metabolite Formation of Butyrylfentanyl by Various Anti-CYP Antibodies
Each value is the average of three measurements.
Fig. 6. Metabolite Formation of Butyrylfentanyl in PXB-Cells
Each value is the average of duplicate measurements.

Vol. 42, No. 4 (2019)
Biol. Pharm. Bull.
629
Fig. 7. Comparison of the Chromatograms for the Monohydroxylated Metabolites of Acetylfentanyl, Fentanyl, and Butyrylfentanyl
Fig. 8. Effect of Hydrolysis on the Detected Amount of the Butyrylfentanyl Metabolites
Each value is the average of three measurements.
butyrylfentanyl, (ω-1)-hydroxy-butyrylfentanyl and β-hydroxy- as 100%. The amounts of 4′-hydroxy-butyrylfentanyl and
butyrylfentanyl) in PXB-cells.To investigate the relationship of 4′-hydroxy-3′-methoxy-butyrylfentanyl drastically decreased
the length of the N-acyl group of fentanyl analogs with their when the hydrolysis step was omitted, indicating that most
metabolite formation patterns, the EICs for the monohydroxyl- of these metabolites were conjugated with glucuronic acid or
ated metabolites of acetylfentanyl, fentanyl, and butyrylfen- sulfate. The amount slightly decreased in case of β-hydroxy-
tanyl were compared (Fig. 7, data for acetylfentanyl and fen- butyrylfentanyl, however, it was not sure whether this me-
tanyl are from our previous study10)). The peaks indicated by tabolite was conjugated or not. A significant decrease was not
arrows are the metabolites hydroxylated at the N-acyl group. observed for the other metabolites, which are not suitable for
Figure 7 clearly indicates that the longer the N-acyl group conjugation. These results are consistent with the results for
(acetyl <propionyl<butyryl), the more amount of N-acyl fentanyl and acetylfentanyl.10)
group-hydroxylated metabolites were formed.
The hydroxylated and carboxylated metabolites of bu-
tyrylfentanyl have the potential to be conjugated. To clarify
whether these metabolites are conjugated, the amount of
CONCLUSION
In this study, the metabolism of butyrylfentanyl was in-
each metabolite in the hydrolyzed culture medium was com- vestigated using fresh human hepatocytes, and the putative
pared with that in the untreated medium. Figure 8 shows the metabolites were chemically synthesized. Seven metabolites
relative amount of each metabolite, where the amount of each of butyrylfentanyl were identified using authentic standards.
metabolite in the hydrolyzed culture medium is expressed In particular, ω-hydroxy- and (ω-1)-hydroxy-metabolites of

630
Biol. Pharm. Bull.
(2008).
Vol. 42, No. 4 (2019)
butyrylfentanyl were definitively identified in our study; these
metabolites were hardly distinguished only by their mass
spectra. In addition, the authentic standards of the metabolites
made accurate quantification possible. Furthermore, it was
revealed that CYP3A4 and CYP2D6 played a key role in the
metabolism of butyrylfentanyl.
4) Drug Enforcement Administration, Department of Justice. Sched-
ules of controlled substances: temporary placement of butyryl
fentanyl and beta-hydroxythiofentanyl into Schedule I. Final order.
Fed. Regist., 81, 29492–29496 (2016).
5) Steuer AE, Williner E, Staeheli SN, Kraemer T. Studies on the
metabolism of the fentanyl-derived designer drug butyrfentanyl
in human in vitro liver preparations and authentic human samples
using liquid chromatography-high resolution-mass spectrometry
(LC-HR-MS). Drug Test. Anal., 9, 1085–1092 (2017).
6) Staeheli SN, Baumgartner MR, Gauthier S, Gascho D, Jarmer J,
Kraemer T, Steuer AE. Time-dependent postmortem redistribution
of butyrfentanyl and its metabolites in blood and alternative matri-
ces in a case of butyrfentanyl intoxication. Forensic Sci. Int., 266,
170–177 (2016).
Acknowledgment This work was supported by Japan
Society for the Promotion of Science (JSPS) KAKENHI Grant
Number 15K08895.
Conflict of Interest The authors declare no conflict of
interest.
7) Yamasaki C, Kataoka M, Kato Y, Kakuni M, Usuda S, Ohzone Y,
Matsuda S, Adachi Y, Ninomiya S, Itamoto T, Asahara T, Yoshizato
K, Tateno C. In vitro evaluation of cytochrome P450 and glucuroni-
dation activities in hepatocytes isolated from liver-humanized mice.
Drug Metab. Pharmacokinet., 25, 539–550 (2010).
REFERENCES
1) United Nations Office on Drugs and Crime. “Fentanyl and its
analogues—50 years on. Global Smart Update, Vol.17, 2017.”:
‹http://www.unodc.org/documents/scientific/Global_SMART_Update_
17_web.pdf›, cited 10 August, 2018.
2) McIntyre IM, Trochta A, Gary RD, Wright J, Mena O. An acute
butyr-fentanyl fatality: a case report with postmortem concentra-
tions. J. Anal. Toxicol., 40, 162–166 (2016).
3) Higashikawa Y, Suzuki S. Studies on 1-(2-phenethyl)-4-(N-
propionylanilino)piperidine (fentanyl) and its related compounds.
VI. Structure–analgesic activity relationship for fentanyl, methyl-
substituted fentanyls and other analogues. Forensic Toxicol., 26, 1–5
8) Siegfried. “Synthesis of fentanyl.”: ‹https://erowid.org/archive/rho-
dium/chemistry/fentanyl.html›, cited 10 August, 2018.
9) Crossland RK, Servis KL. Facile synthesis of methanesulfonate
esters. J. Org. Chem., 35, 3195–3196 (1970).
10) Kanamori T, Togawa-Iwata Y, Segawa H, Yamamuro T, Kuwayama
K, Tsujikawa K, Inoue H. Use of hepatocytes isolated from a liver-
humanized mouse for studies on the metabolism of drugs: applica-
tion to the metabolism of fentanyl and acetylfentanyl. Forensic
Toxicol., 36, 467–475 (2018).