Article

pubs.acs.org/crt

Street-Like Synthesis of Krokodil Results in the Formation of an Enlarged

Cluster of Known and New Morphinans
José Xavier Soares,*,†,‡‡ Emanuele Amorim Alves,*,‡,§,∥,⊥,‡‡ André M. N. Silva,#
Natália Guimaraẽ s de Figueiredo,∇ Joaõ F. Neves,○ Sara Manuela Cravo,○ Maria Rangel,◆
Annibal Duarte Pereira Netto,¶ Félix Carvalho,*,‡ Ricardo Jorge Dinis-Oliveira,*,‡,§,⊥,‡‡
and Carlos Manuel Afonso*,○,††,‡‡
†
LAQV, REQUIMTE, Department of Chemical Sciences, Laboratory of Applied Chemistry, Faculty of Pharmacy, University of Porto,
José Viterbo Ferreira Street No. 228, 4050-313 Porto, Portugal
‡
UCIBIO, REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto,
José Viterbo Ferreira Street No. 228 4050-313 Porto, Portugal
§
Department of Public Health and Forensic Sciences, and Medical Education, Faculty of Medicine, University of Porto,
Prof. Hernâni Monteiro Alameda, 4200-319 Porto, Portugal
∥
EPSJV−Polytechnic School of Health Joaquim Venâncio, Oswaldo Cruz Foundation, Brazil 4.365 Avenue, Manguinhos,
21.040-900 Rio de Janeiro, Brazil
⊥
IINFACTS-Institute of Research and Advanced Training in Health Sciences and Technologies, Department of Sciences,
University Institute of Health Sciences (IUCS), CESPU, CRL, Central de Gandra Street, 1317, 4585-116 Gandra, Portugal
#
LAQV, REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Campo Alegre Street,
4169-007 Porto, Portugal
∇
Laboratory of Tobacco and Derivatives, Analytical Chemistry Division, National Institute of Technology, Venezuela Avenue, 82,
Praça Mauá, 20081-312 Rio de Janeiro, Brazil
○
Department of Chemical Sciences, Laboratory of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, University of Porto,
José Viterbo Ferreira Stree No. 228, 4050-313 Porto, Portugal
◆
LAQV, REQUIMTE, Institute of Science Abel Salazar, University of Porto, José Viterbo Ferreira Street No. 228, 4050-313 Porto, Portugal
¶
Department of Analytical Chemistry, Chemistry Institute, Fluminense Federal University, Outeiro de São João Batista, Valonguinho
Campus, Centro, Niterói, 24020-150, Rio de Janeiro, Brazil
††
Interdisciplinary Center of Marine and Environmental Investigation (CIIMAR/CIMAR), General Norton de Matos Avenue,
4450-208 Matosinhos, Portugal
*
S Supporting Information

ABSTRACT: “Krokodil” is the street name for a homemade injectable drug

that has been used as a cheap substitute for heroin. Codeine is the opioid start-
ing material for krokodil synthesis, and desomorphine is claimed to be
the main opioid of krokodil and the main component responsible for
its addictive and psychoactive characteristics. However, due to its peculiar
manufacture, using cheap raw materials, krokodil is composed of a large and
complex mixture of different substances. In order to shed some light upon the
chemical complexity of krokodil, its profiling was conducted by reverse phase
high performance liquid chromatography coupled to a photodiode array detector
(RP-HPLC-DAD) and by liquid chromatography coupled to high resolution
tandem mass spectrometry (LC-ESI-IT-Orbitrap-MS). Besides desomorphine,
codeine, and morphine, profiting from the high resolution mass spectrometry
(HRMS) data, an endeavor to study the morphinans content in krokodil was
set for the first time. Considering codeine as the only morphinan precursor
and the possible chemical transformations that can occur during krokodil synthesis, the morphinan chemical space was designed,
and 95 compounds were defined. By making use of the morphinan chemical space in krokodil, the exact masses featured by
HRMS, and the morphinan mass fragmentations patterns, a targeted identification approach was designed and implemented.
continued...

Received: May 11, 2017

Published: July 14, 2017

© 2017 American Chemical Society 1609 DOI: 10.1021/acs.chemrestox.7b00126

Chem. Res. Toxicol. 2017, 30, 1609−1621
Chemical Research in Toxicology
The proposed 95 morphinans were searched using the full scan chromatogram of krokodil, and findings were validated by mass
fragmentation of the correspondent precursor ions (MS2 spectra). Following this effort, a total of 54 morphinans were detected,
highlighting the fact that these additional morphinans may contribute to the psychotropic effects of krokodil.
■ INTRODUCTION
“Krokodil” is a complex psychotropic drug mixture with iodine
odor resulting from a homemade synthesis process using easy
access starting materials and is used as a cheap substitute for
heroin.1−9 Krokodil first appeared around 2002/3 in Siberia, and
its use spread throughout other Russian areas and the former
Soviet Republic countries and more recently across Europe,
namely, Germany, Czech Republic, France, Belgium, Sweden,
Norway, Poland, Spain, and Portugal, and also the USA.9−11
Desomorphine is the semisynthetic opioid claimed to be the
main component of krokodil and considered to be responsible
for its addictive and psychoactive effects. However, due to its
nature, the synthesis produces not only desomorphine but also
other side-products as previously described in krokodil samples
and biological fluids of people who inject krokodil (PWIK).2,12
Several toxic effects have been reported and claimed to be related
to impurities, namely, jaw osteonecrosis presenting as alveolar
process exposure in the oral cavity, infections by HIV and
hepatitis A, B, and C, skin and venous damage, including ulcers,
abscesses, scaly and rough phlebitis, like crocodile skin around
the injection sites, gangrene, and limb amputations.1−9 Recently,
we observed that repeated subcutaneous administrations of
krokodil causes skin necrosis and toxicity to internal organs
in Wistar rats, leading to cardiac and renal dysfunction. The
impurities, particularly the high phosphorus concentrations of
krokodil batches, may explain many of the signs and symptoms
present in PWIK.13
Besides pleasure, clinical cases related to PWIK and also
animals exposed to krokodil suggest high pain tolerance to the
corrosive and necrotizing effects.13,14 Therefore, we hypothe-
sized that, besides desomorphine, krokodil may uncover new
morphinan derivatives capable of relieving moderate and severe
pain and contribute to the addictive effects. Aiming to identify
additional morphinans (as a representative skeleton of a large
class of opioids), a suitable analytical methodology, capable
of identifying complex mixtures is required. A great variety of
separation techniques coupled to mass spectrometry (MS) have
been used for the identification of different compounds in clinical
and forensic toxicology due to its capacity to generate information
both on the molecular mass and the structure of molecules.15−22
With the advent of high resolution mass spectrometry (HRMS),
exact masses could be determined, and new perspectives were
opened for liquid chromatography (LC)-HRMS allowing the
study of complex mixtures. Since its first description and its
commercial introduction in 2005, the Orbitrap mass analyzer has
demonstrated great sensitivity and high resolving power.23−25
This high-resolution mass analyzer is particularly attractive due
to its great sensitivity and scan rate, which is ideal for online
coupling with liquid chromatography separation. Indeed,
coupling LC to Orbitrap mass spectrometers equipped with an
electrospray ionization source (ESI) has been providing an
analytical instrument of great utility in different areas of analytical
and forensic chemistry.15,26 Therefore, the use of LC-HRMS
data analysis with the aid of informatic tools, such as Mass
Frontier and Compound Discoverer, may be considered of great
value for the identification of substances in krokodil.27 Therefore,
the goals of this work were to contribute a better knowledge
of krokodil providing new insights into the chemical profile and
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detecting the different morphinans present in krokodil. Morphinans
were identified by comparison with reference standards
(desomorphine, codeine, and morphine) and by making use
of exact masses and mass fragmentations provided by
LC-ESI-IT-Orbitrap-MS.
■ MATERIALS AND METHODS
Reagents and Standards. For krokodil synthesis, gasoline, alkali
solutions for cleaning pipes, and matchboxes were purchased from local
retail stores in Porto, Portugal. Hydrochloric acid 37% was purchased
from VWR Prolabo. Codeine-containing capsules, iodine tinctures,
hydrogen peroxide, and commercial 96% ethanol were purchased from
local pharmacies in Porto, Portugal. For high performance liquid
chromatography (HPLC) analysis, Chromasolv acetonitrile and
LC-MS grade formic acid were obtained from Sigma-Aldrich. Ultrapure
(Milli-Q) water was used throughout the study.
Krokodil Samples. All of the krokodil samples were produced by
street-like synthesis as previous described.28 The synthesis procedure
was repeated 10 times, and a representative pool of the obtained
products was used for further analysis.
Reversed Phase High Performance Liquid Chromatography
with Photodiode Array Detection (RP-HPLC-DAD) Conditions.
For photodiode array detection (DAD) analysis, a Dionex Ultimate
3000 HPLC Basic system (Thermo Scientific), equipped with an
autosampler and a DAD detector, was used. For chromatographic
separation, a C18 Hypersil GOLD with 1.9 μm particle size, 50 mm L,
and 2.1 mm ID (Thermo Scientific) equipped with a C18 BEH
VanGuard (5 mm L × 2.1 mmID) (Waters) guard column was used.
Column temperature was set to 40 °C. Eluent A was aqueous formic acid
(1%), and eluent B was formic acid (1%) in acetonitrile. Samples
(10 μL) were injected directly into the column and washed for 5 min
with an isocratic flux of 98% eluent A and 2% eluent B at a flow rate of
300 μL/min. Subsequent to the initial 5 min wash, a 30 min linear
gradient from 2% to 40% buffer B was applied. The eluent composition
was then raised to 100% buffer B over 15 min and thus maintained for
an 10 additional min in order to allow column cleaning. The column was
re-equilibrated in 98% buffer A/2% buffer B before any further analysis.
DAD spectra were acquired for the full length of the chromatographic
run between 200 and 600 nm. Chromeleon 7.1 SR2 software by Thermo
Fisher Scientific was used to manage chromatographic data. Prior to use,
mobile phase solvents were degassed in an ultrasonic bath for 15 min.
The identification of desomorphine, codeine, and morphine was
established based on the comparison with standard retention time
under the same chromatographic conditions and the UV/vis spectrum
and by coelution.
Reversed Phase High Performance Liquid Chromatography
with Electrospray Ionization Coupled to High Resolution
Tandem Mass Spectrometry Detection Conditions. Krokodil
samples were analyzed by LC-ESI-IT-Orbitrap-MS. Before analysis, the
full krokodil reaction mixture was diluted 10 times in the initial eluent
composition. An Accela 600 HPLC system (Thermo Scientific, Bremen,
Germany), equipped with an autosampler, was coupled online to a
LTQ-Orbitrap XL mass spectrometer (Thermo Scientific, Bremen,
Germany) operated with an ESI source. For chromatographic
separation, a C18 Hypersil GOLD with 1.9 μm particle size, 50 mm L,
and 2.1 mm ID (Thermo Scientific) equipped with a C18 BEH
VanGuard (5 mm L × 2.1 mmID) (Waters) guard column was used.
Column temperature was set to 40 °C. Eluent A was aqueous formic acid
(1%), and eluent B was formic acid (1%) in acetonitrile. Samples
(10 μL) were injected directly into the column and washed for 5 min
with an isocratic flux of 98% eluent A and 2% eluent B at a flow rate of
300 μL/min. Subsequent to the initial 5 min wash, a 30 min linear
gradient from 2% to 40% buffer B was applied. The eluent composition
was then raised to 100% buffer B over 15 min and maintained for
10 additional minutes in order to allow column cleaning. The column
DOI: 10.1021/acs.chemrestox.7b00126
Chem. Res. Toxicol. 2017, 30, 1609−1621
Chemical Research in Toxicology
was re-equilibrated in 98% buffer A/2% buffer B before any further
analysis.
The mass spectrometer was operated in the positive ion mode, with a
spray voltage of 3.1 kV and a transfer capillary temperature of 275 °C.
Sheath and auxiliary gas (nitrogen) flows were set to 40 and 10 (arbitrary
units), respectively. Tube lens voltage was set to 120 V. MS survey scans
were acquired at an Orbitrap resolution of 60000 for an m/z range from
100 to 1000. Tandem MS (MS/MS) data were acquired either in the
linear ion trap or in the Orbitrap at a resolution a 7500. A data
dependent method with dynamic exclusion was employed for MS2
acquisition: the top 3 most intense ions were selected for collision
induced dissociation (CID). CID settings were 50% normalized
collision energy, 2 Da isolation window, 30 ms activation time, and an
activation Q of 0.250. A window of 45 s was used for dynamic exclusion.
Automatic gain control was enabled, and target values were 1.00e6 for
the Orbitrap and 1.00e4 for LTQ MSn analysis. The identification of
desomorphine, codeine, and morphine was established based on the
comparison with standard retention time under the same chromato-
graphic conditions and mass fragmentation spectra (MS2).
Data Acquisition and Processing. HPLC-DAD data were
recorded with Chromeleon 7.1 SR2 software, and Thermo Fisher
Scientific managed the chromatographic data (LC-ESI-IT-Orbitrap-MS
with Xcalibur software version 2.1). The acquired data set from the
injection of four similar samples was processed using Compound
Discoverer 2.0.0.303 with the following nodes: select spectra (default values);
filter centroids (S/N threshold = 3; minimum peak intensity = 1000);
align retention times (default values); a generate expected compound
node for each parent compound (apply dealkylation = false; apply
dearylation = false; phase I = none; phase II = none; ions = [M + H]+1,
[M + Na]+1, [M+NH4]+1; remaining were set to default values); find
expected compounds (mass tolerance = 5 ppm; minimum peak intensity
= 100000; minimum number of isotopes = 2; remaining were set to
default values); group expected compounds (default values); merge
features (retention time tolerance: 0.5 min; remaining were set to
default values); and FISH scoring (default values). Spectra were
searched against an internal database of 95 morphinans attained from
the krokodil chemical space (Table S1). Further analysis of fragment
spectra was performed with Mass Frontier version 7.0.5.9. Retention
Figure 1. Krokodil chromatograms obtained by different detection methodologies. (A) RP-HPLC-DAD in 3D wavelength detection; (B) full MS scan
chromatograms (TIC) of crude krokodil (black line); (C) chromatogram of morphine (RT = 1.96), codeine (RT = 8.21), and desomorphine (RT =
10.41) standards.
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times were selected considering the lower ΔMass (ppm). Morphinan
relative abundance (percentage) was calculated dividing the respective
peak area by the peak areas sum of all detected morphinans. Manual
inspections of chromatograms and mass spectra were conducted with
Xcalibur, version 2.1. Chemical structures were drawn with ChemDraw
Professional, version 16.0.0.82.
■
RESULTS AND DISCUSSION
Krokodil Chemical Profiling. Because of the raw materials,
homemade character, and the harsh reaction conditions used in
its manufacturing, krokodil is a very complex mixture, composed
of a great variety of substances with different chemical and
physicochemical characteristics.2,28 Initial studies in krokodil
were previously performed using normal phase HPLC-DAD and
GC-MS methodologies.12,28 Organic extracts of krokodil and
GC derivatization reagents were used in those analyses. In order
to deeply explore the chemical composition of krokodil, several
studies were performed in samples of crude krokodil, starting
with RP-HPLC-DAD analysis. Since no extraction steps or
derivatization reactions were used, the loss of substances during
these procedures was eliminated, and a more complete profile was
possible. The DAD 3D chromatogram is showed in Figure 1A
(260−400 nm). The chromatogram reveals the expected
complexity of krokodil, with several substances eluting along a
wide range of polarities.
Desomorphine has been considered the chemical marker
for the identity of krokodil.2 However, other morphinans
(i.e., 3,6-dideoxy-dihydromorphine and 4,5-epoxymorphinan-3-ol)
have been found in krokodil samples, which may play an
important role in the additive and psychotropic effects.12,28
Consequently, in order to better understand the krokodil
morphinan composition a LC-ESI-IT-Orbitrap-MS analysis
was conducted, using the optimized RP-HPLC-DAD conditions.
Morphinans are alkaloid derivatives with basic nitrogen making
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Chemical Research in Toxicology
them suitable for ESI analysis in positive mode.29 LC-ESI-IT-
Orbitrap-MS is able to couple the power of chromatographic
separation to the power of MS structure elucidation and
resolution with accurate masses.30 A total ion current (TIC)
chromatogram of crude krokodil in positive mode is showed in
Figure 1B. This provided another perspective over the complex
krokodil profile, revealing the compounds that can be ESI
positive ionizable compounds, such as morphinans. Desomor-
phine, codeine, and, morphine were identified in the crude
krokodil by comparison with retention times, exact masses of
the precursor ions, and mass fragmentation patterns of
standards analyzed under the same chromatographic con-
ditions (Figure 1C; Table 1). Additionally, the identification of
desomorphine, codeine, and morphine was established by
co-injection with the reference standards. As far as we known,
morphine is reported for the first time in krokodil.
Furthermore, the understanding of the fragmentation behavior
of morphinans in the analytical conditions used in this work is
important for establishing morphinan fragmentation patterns.
Therefore, the mass fragmentation spectra of desomorphine,
codeine, and morphine were interpreted (Figure 2) using Mass
Frontier and manual interpretation. Mass Frontier uses a set of
general fragmentation rules and a fragmentation library to
predict the structure of the different fragments.31 Manual
interpretation of spectra was conducted accordingly with mass
fragmentation rules and with morphine and codeine fragmenta-
tion pathways described in the literature.28,31−33 The three
morphinans showed a similar fragmentation behavior. All three
show the piperidine ring loss, leading to the base peak (m/z 215)
in desomorphine and to the intense peaks in codeine (m/z 243)
and morphine (m/z 229). Other typical fragmentation behaviors
were observed. When a α,β-unsaturated hydroxyl moiety is
present, like in morphine and codeine, base peaks are produced
by losing the piperidine ring and CO, leading, respectively, to
fragments m/z 201 and m/z 215.32 In addition, when an alcohol
hydroxyl group is present in ring C, a loss of water was observed,
leading to intense peaks.32 When a phenolic hydroxyl group is
present in ring A and an alcohol hydroxyl group is not present in
ring C, the loss of water was verified leading to a medium
Table 1. Unequivocal Identification of Morphine, Codeine, and Desomorphine
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intensity peak, like in desomorphine (m/z 197). Moreover, the
loss of H2 from different fragments was also a very common
feature, like in desomorphine m/z 213.32
Targeted Detection of Morphinans in Krokodil. The
identification of other morphinans could represent a pivotal
importance to fully understand the psychotropic effects of
krokodil, but it is even more interesting to explain the apparent
profound analgesia to tolerate the injection of a corrosive and
caustic formulation (pH < 1).2,33 It is clear that all morphinans
present in krokodil have to be originated from codeine, the only
possible precursor of this class of compounds present in the
starting materials. From the codeine precursor, three types of
conditions can be outlined in eight different chemical trans-
formations (Figure 3a−i): (a) The codeine to desomorphine
synthetic pathway; three different reactions are involved (a, b,
and c), dehydroxylation at position number 6, reduction of
double bond between positions 7 and 8, and O-demethylation of
the methoxyl group at position 3; (b) the previously reported
desomorphine byproducts, dehydroxylation at position number
3 and N-demethylation (d,e);12,28 (c) codeine byproducts;
considering the highly reductive environment of krokodil
manufacture,28 it is reasonable to assume that a 4,5-epoxy
cleavage can also occur as is the case with the already described
methoxyl cleavage at position 3. The epoxy cleavage will lead to
two possible types of products, a phenol bearing an hydroxyl
group at position 4 or an alcohol at position 5 (f,g). The phenol
obtained from epoxy cleavage products can be further
dehydroxylated (h) as is the case with the already described
dehydroxilation at position 3 (d). The alcohol obtained from
epoxy cleavage products can be further dehydroxylated (i) as is the
case with the already described dehydroxilation at position 6 (a).
Considering these logical and reasoned chemical trans-
formations, it is reasonable to hypothesize that they can occur
in similar ways with all morphinan based compounds present in
krokodil, as long as the necessary chemical conditions are
present. Figure 4 depicts one way to achieve the prototypic
morphinan structure molecule from codeine. Bearing this in
mind and combining the considered chemical transformations of
codeine and derivatives, a total of 95 different compounds can be
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Chemical Research in Toxicology
Figure 2. MS2 fragmentation spectra and fragmentation pathway of desomorphine, codeine, and morphine. Morphinan rings A to E are provided in red.
rationally produced and represent the morphinans chemical
space in krokodil (Table S1). Because of the homemade
uncontrolled production, the probability of the formation of
the hypothesized morphinans and their relative amounts can
be variable. Nevertheless, chemical transformation occurring
on labile positions is certainly favored. It is also noteworthy
that several of the proposed morphinans are constitutional
isomers (same molecular formula, different atoms’ connectivity).
As a consequence of the presence of isomers, from 95 admitted
compounds 30 molecular formulas are possible to establish. The
above-mentioned chemical space can be subdivided into three
categories: (i) morphinans that have already been identified in
krokodil;2,12,22 (ii) morphinans that have not been identified in
krokodil, but structures have been reported in other contexts;
and (iii) morphinans that, as far as we know, have never been
reported at all.
In order to put in evidence the morphinans that are a part of
krokodil, a two-level identification strategy was implemented.
The first level of identification consisted in finding in the MS TIC
chromatogram of krokodil the 95 proposed morphinans. For
this, Compound Discover was used. First, a library with the exact
masses of the proposed morphinans was built, and a screening on
krokodil MS full scan chromatogram for these exact masses was
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performed by the software (Table S1). A hit was defined when
the exact masses of the precursor ion were compatible with the
exact mass of a molecular formula proposed in the library. In this
matching analysis, beyond finding the same exact masses, for hit
validation two additionally acceptance criteria were also
considered: compatibility of the isotopic peak mass ([M + H]+1)
and its relative abundance, and repeatability, which was evaluated
by analyzing four replicates, considering only hits those that
matched in at least three.
The second level of identification was focused in analyzing the
mass fragmentation of each hit precursor ion (MS2 spectra).
Taking into account the similarity of the already discussed
fragmentation pathways of desomorphine, codeine, and
morphine (Figure 5, bold lines), it is reasonable to assume that
other morphinans with similar structural features will present
similar fragmentation behaviors. Dashed lines depict some
representative examples of such morphinans and their respective
fragmentation pathways, showing the characteristic neutral losses
of the piperidine ring (−CH2CHNHCH3, Δm/z = 57.06), water
(-H2O, Δm/z = 18.01) or hydrogen (-H2, Δm/z = 2.01),
as happens with desomorphine, codeine, and morphine.
Accordingly, other morphinans will yield fragments with m/z
values that are dependent on parent ion chemical structure.
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Figure 3. Krokodil chemical space. The inner circle summarizes the proposed chemical transformations involved in krokodil manufacture. Outer circles
provide the rationale that justifies each chemical transformation. (a) dihydroxylation at C6; (b) reduction of C7-C8 double bond; (c) O-demethylation at
C3; (d) dihydroxylation at C3; (e) N-demethylation; (f) ether cleavage at C4; (g) ether cleavage at C5; h) dihydroxylation at C5; i) dehydroxylation at C4.

Consequently, at this second level of identification, hits
were admitted as morphinans if they showed a morphinan
compatible fragmentation behavior. Finally, only validated
hits that fulfill both identification level requirements were
definitely considered as morphinans. Morphinans detected in
krokodil samples are summarized in Table 2. Morphinans were
identified with high accuracy (Δm/z = −1.89−2.81 ppm)
and similar to what is reported for other Orbitrap-based
methods.33−35 In addition, for each validated hit, the mass of
the three most abundant fragments obtained from MS2 frag-
mentation are reported. These fragments were considered in order
to fulfill the requirements of EU Decision (implementing, 2002)
1614
concerning the performance of analytical methods and the
interpretation of results.30
Desomorphine, codeine, and morphine which were already
identified by coelution with standards as previously described
were also put in evidence using this approach. Other 51 morphinans
were detected, making a total of 54 morphinans revealed (Table 2),
in the total of 95 possible morphinans that could be formed
in krokodil. For each morphinan, the molecular formula and the
compatible chemical structure are provided (Table 3). For six of
the detected morphinans, only one compatible structure can be
written, according to our reasoning and the consequently
proposed chemical space (Table S1). For the remaining 40 three
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Chemical Research in Toxicology
Figure 4. One representative synthetic pathway from codeine to morphinan. (a) dehydroxylation; (b) reduction; (c) O-demethylation; (d) dehydroxylation;
(e) N-demethylation; (f) ether cleavage; (h) dehydroxylation. Gray arrows represent the other chemical transformations that could occur for each product.
Figure 5. Morphinan fragmentation pattern. Fragments obtained from codeine, morphine, and desomorphine are represented with solid lines. Dashed
lines are used to indicate the fragmentation of other morphinans.
morphinans, with different retention times, some share the same
molecular formula (isomers).
Codeine, the starting material of krokodil, and morphine were
found in trace amounts, corresponding each one to 1% of the
total morphinans. Desomorphine is the most abundant
morphinan present in krokodil (Table 2, RT = 10.03),
corresponding to near 45%. The second most abundant
morphinan (Table 2, RT = 17.10, C17H21NO), close to 14%,
corresponds to one of the six proposed compounds. It is
reasonable to admit that this morphinan could correspond to
3,6-dideoxy-dihydromorphine since it was already found at
a similar concentration in krokodil.12,28 The majority of
morphinans correspond to a cluster, forming a complex mixture.
■
CONCLUSIONS
Built on top of our previous reports,28,33 the data presented here,
resulting from RP-HPLC-DAD analysis and MS TIC analysis by
LC-ESI-IT-Orbitrap-MS, provided new insights about the
chemical profile of crude krokodil. Because of its chemical
complexity, krokodil analytical characterization is challenging.
In this work, a representative sample that resulted from 10 street-
mimetic syntheses was used. However, according to our experience,
the krokodil composition being always very complex can greatly
vary depending on the manufacture/synthesis conditions.
Desomorphine, codeine, and morphine were detected and
confirmed by co-injection with reference standards, morphine
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being identified for the first time in krokodil. Assuming that
codeine is the only possible morphinan precursor and that possible
chemical transformations followed the rationale behind our
reaction mechanism proposal, a total of 95 morphinans were
defined.28 This chemical space is the cornerstone of a targeted
identification approach based on the exact masses feature and mass
fragmentations patterns provided by LC-ESI-IT-Orbitrap-MS.
In this approach, the 95 proposed morphinans were searched
using the MS TIC chromatogram of krokodil, and findings were
validated by mass fragmentation of the correspondent precursor
ions (MS2 spectra). Following this strategy, 54 morphinans were
detected.
Summarizing the work done so far, it is plausible to hypothesize
that the high psychoactive and analgesic effects of krokodil cannot
be explained only by the presence of desomorphine, the so far
claimed chemical marker of krokodil. Indeed, even performed
under controlled conditions (e.g., same laboratory and operator),
composition varies between batches. While desomorphine is
the main opioid in some batches, in others it is present in lower
amounts. This variability is certainly reflected in real samples of
krokodil. The presence of other morphinans might additively or
synergistically maximize the pharmacokinetics and/or pharma-
codynamics of opioids, but that needs to be further clarified.
PWIK and simultaneous producers of krokodil may have offered,
even in a totally accidental way, an analgesic new therapeutic
arsenal that is truly incomparable. In our opinion, it might be the
source of new therapeutic molecules, although the proof of
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Table 2. Exact Mass and Mass Fragmentation of the Morphinans Identified in Krokodil
RT fwhm % area of isotopic precursor ion error number of Frag. 1 Frag. 2 Frag. 3
(min) (min) morphinans formula mass m/z (ppm) isotopes m/z m/z m/z
22.61 0.13 0.59 C16H19N 225.15175 226.15935 −1.42 4 211.09 198.05 166.94
25.72 0.14 0.79 C16H21N 227.16740 228.17484 −0.70 4 184.00 185.02 186.05
22.29 0.13 1.43 C17H21N 239.16740 240.17496 −1.17 3 225.06 199.06 212.10
17.35 0.15 2.19 C16H19NO 241.14666 242.15424 −1.22 4 197.02 199.01 225.04
14.54 0.10 0.19 C16H19NO 241.14666 242.15399 −0.21 3 199.01 183.98 225.07
13.45 0.07 0.07 C16H19NO 241.14666 242.15405 −0.46 3 199.04 197.02 156.96
22.12 0.15 0.54 C17H23N 241.18305 242.19064 −1.31 3 185.04 128.92 156.98
15.01 0.10 0.13 C16H21NO 243.16231 244.16962 −0.11 3 201.03 199.02 227.03
17.10 0.18 14.02 C17H21NO 255.16231 256.16928 1.21 4 197.01 198.98 181.00
20.29 0.13 0.05 C17H21NO 255.16231 256.16995 −1.41 2 211.03 213.04 225.06
19.15 0.10 0.12 C17H21NO 255.16231 256.16983 −0.93 3 199.02 225.01 197.04
15.28 0.11 0.52 C17H21NO 255.16231 256.16974 −0.58 4 198.99 196.95 225.02
21.17 0.10 0.12 C17H21NO 255.16231 256.16983 −0.93 3 211.04 213.05 199.02
12.84 0.09 0.37 C17H21NO 255.16231 256.17007 −1.89 3 199.00 197.02 156.95
12.81 0.11 0.23 C16H19NO2 257.14158 258.14896 −0.39 4 215.00 229.04 241.01
9.87 0.07 1.51 C16H19NO2 257.14158 258.14905 −0.75 3 215.03 212.98 197.03
11.54 0.10 0.07 C16H19NO2 257.14158 258.14908 −0.86 3 227.05 215.04 148.90
14.55 0.16 2.55 C17H23NO 257.17796 258.18515 0.35 4 199.02 201.03 227.07
11.60 0.10 0.09 C17H23NO 257.17796 258.18527 −0.12 3 227.05 215.04 148.90
12.69 0.08 0.03 C17H23NO 257.17796 258.18527 −0.12 2 215.00 229.04 241.01
17.27 0.11 0.01 C16H21NO2 259.15723 260.16437 0.53 2 229.08 203.03 185.05
13.64 0.08 0.03 C16H21NO2 259.15723 260.16467 −0.64 2 217.03 214.95 243.09
18.96 0.09 0.03 C16H21NO2 259.15723 260.16476 −1.00 2 203.04 182.98 229.07
5.89 0.12 3.11 C17H19NO2 269.14158 270.14902 −0.60 3 194.99 213.04 192.95
21.18 0.10 0.37 C18H23NO 269.17796 270.18530 −0.23 3 213.03 211.07 195.03
3.62 0.10 0.03 C17H21NO2 271.15723 272.16449 0.06 3 215.04 227.07 216.00
5.06 0.14 0.07 C17H21NO2 271.15723 272.16452 −0.05 3 215.03 197.02 216.01
14.39 0.11 0.40 C17H21NO2 271.15723 272.16458 −0.28 4 215.01 229.04 196.99
6.90 0.28 0.14 C17H21NO2 271.15723 272.16461 −0.39 3 227.11 216.00 214.98
15.66 0.12 2.68 C17H21NO2 271.15723 272.16476 −0.95 4 241.05 215.04 213.04
11.80 0.10 1.87 C17H21NO2 271.15723 272.16479 −1.06 3 215.04 148.89 172.96
19.25 0.10 0.75 C17H21NO2 271.15723 272.16470 −0.73 5 241.00 182.97 223.05
21.53 0.10 0.06 C17H21NO2 271.15723 272.16467 −0.62 2 182.97 223.01 215.98
10.03 0.36 45.46 C17H21NO2 271.15723 272.16473 −0.84 3 215.00 197.00 212.99
12.95 0.12 5.13 C17H21NO2 271.15723 272.16473 −0.84 3 215.01 148.92 194.99
12.34 0.12 6.15 C17H21NO2 271.15723 272.16486 −1.29 3 215.01 229.03 216.01
2.36 0.06 0.04 C16H19NO3 273.13649 274.14377 0.01 3 198.97 231.03 213.04
17.26 0.11 0.16 C17H23NO2 273.17288 274.18024 −0.30 3 148.91 217.01 197.00
15.28 0.10 0.53 C17H23NO2 273.17288 274.18008 0.26 3 217.02 148.93 196.99
11.34 0.09 0.20 C17H23NO2 273.17288 274.18033 −0.63 3 199.00 196.98 225.04
15.15 0.11 0.95 C18H21NO2 283.15723 284.16443 0.27 3 194.98 227.02 192.96
1.87 0.10 1.11 C17H19NO3 286.14389 285.13649 −0.42 3 201.02 229.02 211.02
17.01 0.13 0.11 C18H23NO2 285.17288 286.17935 2.81 2 229.06 211.05 227.01
13.42 0.08 0.22 C18H23NO2 285.17288 286.18018 −0.07 3 229.06 211.07 209.03
20.89 0.10 0.12 C18H23NO2 285.17288 286.18015 0.04 3 229.02 197.04 243.04
5.08 0.18 0.08 C17H21NO3 287.15214 288.15939 0.09 2 213.02 270.11 194.99
4.37 0.13 0.01 C17H21NO3 287.15214 288.15936 0.20 2 212.99 231.01 194.97
1.52 0.07 0.15 C17H21NO3 287.15214 288.15948 −0.22 3 194.99 270.09 213.02
2.92 0.13 2.30 C17H21NO3 287.15214 288.15952 −0.33 2 231.01 199.01 213.00
4.49 0.14 0.24 C17H23NO3 289.16779 290.17505 0.07 3 215.03 213.02 241.05
3.48 0.12 0.23 C17H23NO3 289.16779 290.17514 −0.24 3 215.02 212.99 241.04
8.09 0.18 1.43 C18H21NO3 300.15952 299.15214 −0.32 4 215.04 243.04 282.10
5.38 0.14 0.13 C18H23NO3 301.16779 302.17508 −0.03 3 194.97 227.04 192.91
10.93 0.09 0.04 C18H23NO3 301.16779 302.17505 0.07 3 194.98 226.99 192.99

concept for the activity and potential clinical use of the identified
and especially of the unknown compounds needs to be further
addressed. Previous structure−activity relationship studies
demonstrated that the amine nitrogen (mostly protonated at
physiological pH) is the most important functional group for
1616
opioid receptor affinity since it permits receptor anchoring
through an essential electrostatic bond with the Asp residue of
the μ-receptor conserved in all G protein-coupled receptors.36−38
The basic nitrogen is most often tertiary for optimal activity.39
Since the krokodil synthesis occurs in a highly reductive
DOI: 10.1021/acs.chemrestox.7b00126
Chem. Res. Toxicol. 2017, 30, 1609−1621
Chemical Research in Toxicology Article

Table 3. Formula, Retention Time, and Compatibles Chemical Structures for the Morphinans Identified in Krokodil

1617 DOI: 10.1021/acs.chemrestox.7b00126

Chem. Res. Toxicol. 2017, 30, 1609−1621
Chemical Research in Toxicology Article

Table 3. continued

1618 DOI: 10.1021/acs.chemrestox.7b00126

Chem. Res. Toxicol. 2017, 30, 1609−1621
Chemical Research in Toxicology Article

Table 3. continued

a
Only one chemical structure is compatible with the detected molecular formula. bIdentified by co-injection with reference standards.

environment, chemical transformations mainly lead to the loss of care in the context of terminal neoplasm, polytraumatization,
oxygen-containing functional groups, and therefore, more burns, adjuvant or postsurgery, acute pulmonary edema, and
lipophilic compounds are produced. The phenolic hydroxyl in multiple medical specialties can now benefit from research on
C3 has been claimed to be important for biological activity since these new alternative morphinans that we first described in
the moiety C3−O is part of the pharmacophore. Indeed, the krokodil.
substitution of phenolic hydroxyl in C3 for -H, -OCH3, or
− OCOCH3 reduces opioid activity.39 This can be easily
understood by the fact of that the analgesic activity of codeine
■
*
ASSOCIATED CONTENT
S Supporting Information
can be attributed to the O-demethylation to morphine. The The Supporting Information is available free of charge on the ACS
C6 hydroxyl is believed to engage in a week hydrogen bond with Publications website at DOI: 10.1021/acs.chemrestox.7b00126.
an Asn residue present in receptors.40 Despite this reported
drug−receptor interaction, the loss of the C6 hydroxyl group Library with the proposed 95 morphinans (PDF)
results in an approximate 10-fold increase of activity due to a
significant increase in lipophilicity.40 Finally, if the 7,8 double
bond is reduced, the C ring becomes significantly more flexible,
■ AUTHOR INFORMATION
Corresponding Authors
and opioid activity increases. If the C7−C8 double bond is *(J.X.S.) E-mail: jfxsoares@ff.up.pt.
reduced, the C ring adopts a more stable chair conformation *(E.A.) E-mail: manuhpa@hotmail.com
which promotes the increase of opioid activity.40 Finally, *(F.C.) E-mail: felixdc@ff.up.pt.
morphinan based analogues, without the 4,5-epoxy bridge, *(R.J.D.-O.) E-mail: ricardinis@med.up.pt
C7−C8 double bond, and 6-hydroxyl group, showed stereo- *(C.A.) E-mail: cafonso@ff.up.pt
selective opioid activity.41 In this context, we will further study
whether we can design the “good part of krokodil” through the ORCID
elimination of necrotic impurities that greatly cause morbidity Ricardo Jorge Dinis-Oliveira: 0000-0001-7430-6297
and may even cause death. Refractory therapies with morphine Author Contributions
‡‡
and other opioids, namely, for severe pain of multiple nature/ J.X.S. and E.A.A. contributed equally to this work. F.C., R.J.D.-O.
causes such as acute myocardial infarction, patients in palliative and C.M.A are co-principal investigators of this study.
1619 DOI: 10.1021/acs.chemrestox.7b00126
Chem. Res. Toxicol. 2017, 30, 1609−1621
Chemical Research in Toxicology
Funding
E.A. and A.D.P.N. acknowledge Brazilian agency Conselho
Nacional de Desenvolvimento Cientifico ́ e Tecnológico (CNPq)
(process 245844/2012-0) for research grants and scholarship.
J.S. thanks National Funds from FCT (Fundaçaõ para a Ciência
e a Tecnologia), FEDER under Program PT2020 (project 007265
-UID/QUI/50006/2013), and through the FCT PhD Programmes
and by Programa Operacional Potencial Humano (POCH),
specifically by the BiotechHealth Programme (Doctoral Pro-
gramme on Cellular and Molecular Biotechnology Applied to
Health Sciences), reference PD/00016/2012 for for financial
support. J.S. also thanks FCT and POPH (Programa Operacional
Potencial Humano) for his Ph.D. grant (SFRH/BD/98105/2013).
F.C. received financial support from the European Union (FEDER
funds POCI/01/0145/FEDER/007728) and National Funds
(FCT/MEC, Fundaçaõ para a Ciência e Tecnologia and Ministério
da Educaçaõ e Ciência) under the Partnership Agreement PT2020
UID/MULTI/04378/2013, as well as the project NORTE-01-
0145-FEDER-000024, supported by Norte Portugal Regional
Operational Programme (NORTE 2020), under the PORTUGAL
2020 Partnership Agreement (DESignBIOtecHealth-New Tech-
nologies for three Health Challenges of Modern Societies:
Diabetes, Drug Abuse and Kidney Diseases), through the European
Regional Development Fund (ERDF). R.D.-O. acknowledges FCT
for his Investigator Grant (IF/01147/2013). S.C. and C.A.
acknowledge FCT through the strategic project CEQUIMED-UP
(Pest-OE/SAU/UI4040/2014).
Notes
The authors declare no competing financial interest.
■
ABBREVIATIONS
CID, collision induced dissociation; DAD, photodiode array
detector; ESI, electrospray ionization; HPLC, high performance
liquid chromatography; HRMS, high resolution mass spectrom-
etry; IT, ion trap; LC, liquid chromatography; LC-ESI-IT-
orbitrap-MS, iquid chromatography with electrospray ionization
high resolution tandem mass spectrometry; LTQ, linear trap
quadrupole; MS, mass spectrometry; MS/MS, tandem mass
spectrometry; PWIK, people who inject krokodil; RP-HPLC-
DAD, reversed phase high performance liquid chromatography
with photodiode array detection; RT, retention time; TIC, total
ion current
■
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