Forensic Science International 195 (2010) 68–72

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Forensic Science International

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Studies on 1-(2-phenethyl)-4-(N-propionylanilino)piperidine (fentanyl) and

related compounds
VII. Quantification of a-methylfentanyl metabolites excreted in rat urine
Shinichi Sato a, Shinichi Suzuki b,*, Xiao-Pen Lee a, Keizo Sato a
a
Department of Legal Medicine, Showa University, School of Medicine 1-5-8, Hatagaya, Shinagawa-ku, Tokyo 142-8555, Japan
b
Third Department of Forensic Science, National Research Institute of Police Science 6-3-1, Kashiwanoha, Kashiwa-shi, Chiba-prefecture 277-0882, Japan

A R T I C L E I N F O A B S T R A C T

Article history: The use of chemically modified controlled drugs, called designer drugs, is widespread internationally. In
Received 30 April 2009 the 1980s, the dominant drugs of abuse were modifications of fentanyl formed by methylation of both
Received in revised form 5 November 2009 the a-position of its phenethyl group (a-methylfentanyl) and the 3-position of its piperidine ring (3-
Accepted 17 November 2009
methylfentanyl). Numerous analytical methods for fentanyl and its analogues, and many studies of its
Available online 16 December 2009
metabolism and major metabolites, have been reported. However, minor metabolites that reflected
injection of the original compound were not included in these studies. Recently, structures of four novel
Keywords:
and minor metabolites that reflect a-methylfentanyl have been reported. This study reports excretion
Forensic toxicology
amounts of these compounds for 96 h following peroral injection to rats of 3 mg/day and urine collection
Fentanyl
1-(2-Phenethyl)-4-(N- every 24 h. Major metabolites were the same as for fentanyl, with approximately 24% of a-
propionylanilino)piperidine analogue methylfentanyl excreted as nor-fentanyl and 15% as v, v-1 hydroxypropiony nor-fentanyl up to 72 h
a-Methylfentanyl post-injection. The novel metabolites were completely excreted within 48 h of injection and composed
Metabolites 2–3% of the total metabolite pool. The major metabolite nor-fentanyl was detected up to 72 h after
Designer drug injection.
ß 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction graphy–mass spectrometry (GC–MS) [8,9], and nuclear magnetic

Morphine, cannabis and cocaine are classically abused
controlled drugs. In addition to these, the past two or three
decades have seen the development of chemically synthesized
modifications of controlled drugs, called designer drugs. The use
of designer drugs has become widespread internationally. Of
these compounds, 3,4-methylenedioxy methamphetamine and
its analogues have the highest rates of abuse. However, recently
the use of tryptamine analogues has spread in Japan. New
designer drugs are continually developed around the world in
clandestine laboratories. In the 1980s in the United States, the
most abused designer drugs were fentanyl analogues [1–5]. The
analogues most popular as designer drugs involved methylation
of the a-position of phenethyl group bound to the 1-position of
piperidine ring (a-methylfentanyl) and the 3-position of the
piperidine ring (3-methylfentanyl) [4–6]. Consequently, numer-
ous analytical methods for fentanyl analogues have been
reported including; infrared spectroscopy [7,8], gas chromato-
* Corresponding author. Tel.: +81 4 7135 8001; fax: +81 4 7133 9153.
E-mail address: suzukis@nrips.go.jp (S. Suzuki).
URL: http://www.nrips.go.jp/
resonance spectrometry (NMR) [8]. Furthermore, structure-
analgesic activities of these analogues have also been reported
[10–14]. Numerous studies have investigated the metabolism
and major metabolites of fentanyl [15–20]. However, reports
concerning fentanyl analogues are limited [21]. This study
investigated what metabolites existed that could indicate
injection of a-methylfentanyl (1). The structures of minor novel
metabolites were reported previously, but quantification of
these metabolites excreted in rat urine was not discussed [22].
In this report, ten rats were injected with 3 mg/kg of a-
methylfentanyl, urine was collected every 24 h up to 96 h post-
injection. Urinary excretion of both major and minor metabolites
was investigated, focusing on details of those novel minor
metabolites that reflected the structure of original compound,
a-methylfentanyl. Four minor metabolites involved some
hydroxy modification of the aromatic rings and propionyl group
in the a-methylfentanyl structure. The first one was of the
phenethyl group (p-aromatic hydroxy a-methylfentanyl (2),
while the second and third metabolites were of the v or v-1
position of hydroxylated (v, v-1 hydroxypropionyl a-methyl-
fentanyl, 3 and 4) ones. A fourth novel minor metabolite included
hydroxy modification of both the p-position of aromatic ring of the
phenethyl group and v position of propionyl group hydroxylated

0379-0738/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2009.11.014
 S. Sato et al. / Forensic Science International 195 (2010) 68–72 69

(p-aromatic hydroxyl and v hydroxypropionyl a-methylfentanyl,
5). These novel minor metabolites were identified by comparison
of compounds synthesized [22]. The major metabolites of a-
methylfentanyl were nor-fentanyl (6) and v, v-1 hydroxypro-
pionyl nor-fentanyl (7 and 8); these contributed about 24, 7.3 and
6.8%, respectively, of the metabolized and also excreted a-
methylfentanyl.
Twenty-four hours after peroral injection of a-methylfenta-
nyl, nor-fentanyl was excreted dominantly and v, v-1 hydro-
xypropionyl nor-fentanyl were excreted as major metabolites.
The p-aromatic hydroxy and v or v-1 hydroxypropiony a-
methylfentanyls were detected in urine 24 h after injection, with
p-aromatic hydroxy a-methylfentanyl detected until 48 h post-
injection. The structures of a-methylfentanyl (1), the examined
metabolites of a-methylfentanyl (from 2 to 8) excreted in rat
urine and the presumed metabolic pathway of a-methylfentanyl
are shown in Fig. 1. This experiment was conducted under
permission from the Ethical Committee of Banyu Pharmaceutical
Co. Ltd. (Tokyo, Japan).
2. Materials and methods
2.1. Synthesis of a-methylfentanyl and its metabolites
a-Methylfentanyl was synthesized according to the previously
reported method [6], its metabolites found in rat urine were also
synthesized by a method described earlier [6,9,22–25]. These
authentic metabolites were utilized for constructing calibration
curves for quantitative determination of metabolites excreted in
rat urine. The metabolic pathways for a-methylfentanyl have been
reported beforehand [22], including both major (large arrows) and
minor metabolic pathways (dotted arrows) in Fig. 1.
2.2. Chemicals
Trifluoroacetyl anhydride was purchased from Tokyo Kasei
Industry Co. Ltd. (Tokyo, Japan). The starting compound for
synthesis of all metabolites, 1-carbethoxy-4-piperidone, was
purchased from Aldrich Co. Inc. (St. Louis, MO, USA). Enzymatic
digestion of glucuronized metabolites was conducted using b-
glucuronidase purchased from Sigma Co. Inc. (Milwaukee, WI,
USA). All other reagents obtained were analytical grade. Water
used during the experiment was Milli-Q grade prepared with the
Milli-Q system (Nippon Millipore, Tokyo, Japan).
2.3. Animal, injection, urine collection and enzymatic digestion
Ten male Wister rats (120–150 g) were used in this study. They
were injected with ca.1.5 ml of a 3% a-methylfentanyl oxalic acid
salt aqueous solution. The solution was administrated perorally.
This corresponded to a 3 mg a-methylfentanyl dosage, the LD50
values for this dosage amount had been reported previously [14]
and were taken into consideration. Urine was collected from each
rat every 24 h and the individual samples combined. Sampling was
conducted until 96 h post-injection. The urine collected (combined
approximately 30 ml) each time (0–24, 24–48, 48–72 and 72–
96 h), was pH adjusted to 5.0 with 1 M acetic acid (approximately
10 ml) and 100 mg of fentanyl was added as an internal standard.
These urine samples were treated with b-glucuronidase (500
units/ml, 37 8C, 12 h) for enzymatic digestion of glucuronides of

Fig. 1. The structures of fentanyl and its analogues: 1, a-methylfentanyl; 2, p-aromatic hydroxy a-methylfentanyl; 3, v hydroxypropionyl a-methylfentanyl; 4, v-1
hydroxypropionyl a-methylfentanyl; 5, p-aromatic hydroxy and v hydroxypropionyl a-methylfentanyl; 6, nor-fentanyl; 7, v hydroxypropionyl nor-fentanyl; 8, v-1
hydroxypropionyl nor-fentanyl.and metabolites detected in rat urine after peroral injection of a-methylfentanyl oxalate. *: Major metabolites of a-methylfentanyl. :
Minor metabolites that reflect the original structure of a-methylfentanyl metabolic pathways are indicated by bold arrows (major) and dotted arrows (minor).
70 S. Sato et al. / Forensic Science International 195 (2010) 68–72
Table 1
GC/MS and quantification conditions.
Instrument M-80 sector mass spectrometer (Hitachi Co. Ltd., Tokyo, Japan)
GC condition
Column 1% OV-17/Chromosorb W (AW-DMCS), 100–120 mesh;
Length 2m
Diameter 3 mm
Oven temperature From 130 to 270 8C (5 8C/min), hold 270 8C for 20 min
Carrier gas N2/50 ml/min
MS condition
Ionization method Chemical impact mode
Ionization voltage 110 mA
Reaction Gas iso-Butane
Utilized m/z for quantification of metabolites of a-methylfentanyl
m/z 463
m/z 575
m/z 351
m/z 329
m/z 441
hydroxyl metabolites of a-methylfentanyl, as the same method
previously reported [22].
2.4. Extraction and trifluoroacetyl derivatization
Following enzymatic digestion by b-glucuronidase, the pH of
the digested urine samples was adjusted with 1 M sodium
carbonate aqueous. solution (pH 8 and approximately 10 ml).
After pH adjustment and were extracted three-times with 50 ml of
chloroform–iso-propanol (3:1, v/v). Solvent was then removed
from the combined extracts at 30 8C in a water bath with an
evaporator. The residues were re-dissolved in ethylacetate (2 ml),
and placed in reaction vials, following by removal of ethylacetate
under N2 flow in a heating block at 30 8C. The residue was re-
dissolved in ethylacetate (100 ml) and trifluoroacetic acid anhy-
dride (50 ml) was added. The reaction vial was sealed tightly and
placed in a heating block at 40 8C for 30 min, followed by solvent
removal under N2 flow at 30 8C. The acyl-derivatized residue was
dissolved in ethyl acetate (50 ml), and 1 ml of this solution was
injected to GC–MS. GC–MS conditions are summarized in Table 1.
2.5. Preparation of calibration curves of metabolites of
a-methylfentanyl
Calibration curves of each metabolite were prepared for each
metabolite as following method, 1 mg/ml of stock solution which
containing a-methylfentanyl, its minor metabolites (2–5) and
another stock solution which containing 1 mg/ml of major
metabolites (6–8) and 1 mg/ml of fentanyl stock solution were
prepared and were diluted by water to the adding amounts. To the
three 10 ml of drug-free control rat urine, 10, 50 and 100 mg of
each minor metabolite (2–5) and 100, 200 and 500 mg of
unchanged a-methylfentanyl and each major metabolite (1,6–8)
were added, respectively. Furthermore, 100 mg of fentanyl were
added as an internal standard. These solutions were extracted and
trifluoroacetyl derivatized as same procedure to the a-methyl-
fentanyl administrated rat urine. The area of multiple ion intensity
were measured using following m/z, for minor trifluoroacetylated
metabolites, using m/z 463, [M+H]+ of p-aromatic hydroxy a-
methylfentanyl (2), m/z 463, [M+H]+ of v hydroxypropionyl a-
methylfentanyl (3) and v-1 hydroxypropionyl a-methylfentanyl
(4), m/z 575, [M+H]+ of p-aromatic hydroxy and v hydroxyproionyl
a-methylfentanyl (5), respectively. For preparation of calibration
curves of unchanged a-methylfentanyl (1) and its major trifluor-
oacetylated metabolites, m/z 351, [M+H]+ of a-methylfentanyl (1),
m/z 329, [M+H]+of nor-fentanyl (6), m/z 441, [M+H]+ of v
p-Aromatic hydroxy a-methylfentanyl (2)
v, v-1 hydroxypropionyl a-methylfentanyl (3 and 4)
p-Aromatic hydroxy and v hydroxypropionyl a-methylfentanyl (5)
a-Methylfentanyl (1)
nor-Fentanyl (6)
v, v-1 hydroxypropionyl nor-fentanyl (7 and 8)
hydroxypropionyl nor-fentanyl (7) and v-1 hydroxypropionyl nor-
fentanyl (8), were selected, respectively. and m/z 315 ([M+H]+) of
fentanyl was used for calculating the intensity ratios for a-
methylfentanyl and its metabolites. The calibration curves
prepared by this method showed satisfactory linearity in these
concentrations of each compounds (r2 > 0.95).
2.6. The recovery yield of the metabolites and a-methylfentanyl
by this procedure
The recovery yield was examined by adding 10, 50 and 100 mg
of p-aromatic hydroxy a-methylfentanyl (2), v hydroxypropionyl
a-methylfentanyl (3), v-1 hydroxypropionyl a-methylfentanyl
(4), p-aromatic hydroxy and v hydroxyproionyl a-methylfentanyl
(5) to the 10 ml of drug free-rat urine and extracted by the same
procedure using CHCl3–iso-propanol (3:1, v/v). The evaporated
residue was trifluoroacetylated and 100 mg of fentanyl was added
as an external standard, and diluted to 50 ml by ethylacetate and
1 ml was injected to GC–MS. Another sample urine was prepared
by adding 100, 200 and 500 mg of a-methylfentanyl (1), nor-
fentanyl (6), v hydroxypropionyl nor-fentanyl (7) and v-1
hydroxypropionyl nor-fentanyl (8) to the 10 ml drug free-rat
urine, and extracted by the same procedure using CHCl3–iso-
propanol (3:1, v/v). The evaporated residue was trifluoroacetylated
and 100 mg of fentanyl was added as an external standard and was
introduced to GC–MS. For adding examination, to the residue of
10 ml drug free-rat urine, 10, 50 and 100 mg of p-aromatic hydroxy
a-methylfentanyl (2), v hydroxypropionyl a-methylfentanyl (3),
v-1 hydroxypropionyl a-methylfentanyl (4), p-aromatic hydroxy
and v hydroxyproionyl a-methylfentanyl (5) were added and
extracted, trifluoroacetylated, diluted by ethylacetate and 100 mg
of fentanyl was added as an external standard and injected GC–MS
by the same procedure previously described. The peak area ratios
to fentanyl were calculated and compared with the results of
former extracting experiment. The recovery was 2 (82.6%, 85.5%
and 85.4% in each concentration, respectively), 3 (80.5%, 81.2% and
82,2%), 4 (81.5%, 83.5% and 83.3%) and 5 (68.5% 68.5% and 69.2%).
The recoveries ware unchanged 1(88.6%, 88.5% and 89.5%), 6
(77.8%, 79.3% and 79.5%) 7(73.5%, 72.8% and 72.2%) and 8 (73.3%,
72.6% and 74.5%).
2.7. The lowest limit of detection (LOD) and lowest limit of
quantification (LOQ)
To define lowest detection limits of detection (LOD) and lowest
limit of detection (LOD) are signal-to-noise = 3 and 10, respec-
 S. Sato et al. / Forensic Science International 195 (2010) 68–72 71

Table 2
Excreted in rat urine of unchanged and metabolites of a-methylfentanyl.

Excreted in rat urine of unchanged and metabolites of a-methylfentanyl (%)

Compound no. Time after the injection

0–24 h 24–48 h 48–72 h 72–96 h

a-Methylfentanyl, 1 2.9  0.8 LOQa LODb LOD

p-Aromatic hydroxy a-fentanyl, 2 1.0  0.3 0.3  0.1 LOQ LOD
v-Hydroxypropionyl a-methylfentany, 3 0.1  0.02 LOQ LOD LOD
v-1 Hydroxypropionyl a-methylfentany, 4 0.1  0.03 LOQ LOD LOD
p-Aromatic hydroxy and v hydroxypropionyl a-methylfentany, 5 0.1  0.03 LOQ LOD LOD
nor-Fentanyl, 6 15.0  2.2 6.0  1.3 2.7  1.1 LOQ
v Hydroxypropionyl nor-fentanyl, 7 2.0  1.0 2.6  0.8 1.2  0.4 LOD
v-1 Hydroxypropionyl nor-fentanyl, 8 2.2  0.8 3.2  0.6 1.9  0.4 LOD

The experiment was repeated 3 times, mean  SD.

Intake of a-methylfentanyl is proved by the comparison of the excreted metabolites found in dark area. Compounds numbers are corresponded to compounds described in Fig. 1.
a
LOQ: limit of quantification.
b
LOD: limit of detection.

tively. In this experiment, LOD and LOQ were examined in drug
free-rat urine extracts. The diluted minor and major metabolites
stock solutions to extracts of 10 ml drug-free rat urine, and
trrifluoroacetylated by the method mentioned above, and diluted
50 ml ethylacetate and 1 ml was introduced GC–MS and measured
the signal-to-noise of each compound (1–8) by the utilizing same
m/z for preparation of calibration curves of each metabolite.
3. Results and discussion
3.1. The LOD and LOQ of each metabolite and unchanged
a-methylfentanyl
The LOD and LOQ of the metabolites of a-methylfentanyl and
unchanged a-methylfentanyl were determined by using each
calibration curve prepared. The LOQ of minor metabolites of a-
methylfentanyl (2–5), p-aromatic hydroxy a-methylfentanyl (2)
were about 300 ng/injection and its LOD was about 100 ng/
injection. LOQ and LOD of v and v 1 hydroxypropionyl a-
methylfentanyl (3 and 4) were about 300 ng/ingection and 100 ng/
injection, respectively. But LOQ and LOD of p-aromatic hydroxy
and v hydroxypropionyl a-methylfentanyl (5) were 500 ng for
LOQ and 300 ng for LOD, respectively. Hence, The LOQ and LOD of
a-methylfentanyl (1) was 500 ng and 200 ng/injection. LOD and
LOQ of nor-fentanyl and v, v-1 nor-fentanyl (6–8) were 400 and
200 ng/injection, respectively. This phenomena was considered to
be caused by the interference components of rat urine.
3.2. Excreted metabolites in rat urine 24 h after injection
The percentage contribution of each metabolite to the total
metabolite pool was determined from calibration curves of the
authentic compounds. The major metabolite found in urine
following a-methylfentanyl injection was nor-fentanyl (6, 15%), a
similar result to fentanyl metabolism; this was detected 24 h after
injection. Unchanged a-methylfentanyl (1) was detected and
quantified at 2.9%. Other metabolites were quantified and composed
2.2% (v hydroxypropionyl nor-fentanyl, 7) and 6.8% (v-1 hydro-
xypropionyl nor-fentanyl, 8) of the major metabolite pool.
Metabolites reflecting the structure of a-methylfentanyl were
also quantified; p-aromatic hydroxy a-methylfentanyl (2, 1.0%), v
hydroxypropionyl a-methylfentanyl (3, 0.1%), v-1 hydroxypropio-
nyl a-methylfentanyl (4, 0.1%), and p-aromatic hydroxy and v
hydroxyproionyl a-methylfentanyl (5, 0.1%).
3.3. Excreted metabolites in rat urine 48, 72 and 96 h after injection
These results are shown in Table 2. Between 24 and 48 h after
injection, the concentrations of unchanged a-methylfentanyl (1),
v hydroxypropionyl a-methylfentanyl (3) and v-1 hydroxypro-
pionyl a-methylfentanyl (4) decreased below the limit of
quantification (LOQ, signal-to-noise > 10). Major metabolites,
nor-fentanyl (6, 6.0%), v hydroxypropionyl nor-fentanyl (7,
3.2%), v-1 hydroxypropionyl nor-fentanyl (8, 2.6%), and p-
aromatic hydroxy a-methylfentanyl (2, 0.3%) were detected.
Between 48 and 72 h, only three major metabolites were
quantified, including v hydroxypropionyl nor-fentanyl (7, 1.9%)
and v-1 hydroxypropionyl nor-fentanyl (8, 1.2%). After 72 h, all
metabolites (6–8) of a-methylfentanyl (1) were below the LOQ and
minor metabolites (2–5) were below LOD.
4. Conclusion
Due to synthetic modifications, designer drugs show significant
structural variation. Consequently, it can be difficult to determine
if the original drug was controlled or not, one potentially valuable
method is the examination of minor metabolites that reflect the
original drug structure. To facilitate this, investigations into
metabolites that reflect the original abused drugs are required.
In the metabolism of a-methylfentanyl, the major metabolites,
nor-fentanyl (6), v hydroxypropionyl nor-fentanyl (7) and v-1
hydroxypropionyl nor-fentanyl (8) were found to be identical to
those of fentanyl itself. However, characteristic metabolites, such
as p-aromatic hydroxy a-methylfentanyl (2), v and v-1 hydro-
xypropionyl a-methylfentanyl (3 and 4), and p-aromatic hydroxy
and v hydroxypropionyl a-methylfentanyl (5), were also excreted
in rat urine until 24 h after injection. By the investigation of these
minor metabolites, the dosed drug should be identified as a-
methylfentanyl, not fentanyl.
Acknowledgment
This work was supported in part by Grant-in-Aid for Scientific
Research (No. 21590744) from the Ministry of Education, Science,
Sports and Culture of Japan.
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