Mass spectrometric evaluation of mephedrone in vivo human metabolism: Identification of phase i and phase II metabolites, including a novel succinyl conjugate

Article abstract of DOI:10.1124/dmd.114.061416

In recent years, many new designer drugs have emerged, including the group of cathinone derivatives. One frequently occurring drug is mephedrone; although mephedrone was originally considered as a legal high product, it is currently banned inmostWestern countries. Despite the banning, abuse of the drug and seizures are continuously reported. Although the metabolism of mephedrone has been studied in rats or in vitro using human liver microsomes, to the best of our knowledge, no dedicated study with human volunteers has been performed for studying the in vivo metabolism of mephedrone in humans. Therefore, the aim of this study was to establish the actual human metabolism of mephedrone and to compare it with other models. For this purpose, urine samples of two healthy volunteers, who ingested 200 mg mephedrone orally, were taken before administration and 4 hours after substance intake. The discovery and identification of the phase I and phase II metabolites of mephedrone were based on ultra-high-performance liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry, operating in the so-called MS^E mode. Six phase I metabolites and four phase II metabolites were identified, four of them not previously reported in the literature. The structure of four of the detected metabolites was confirmed by synthesis of the suggested compounds. Remarkably, a mephedrone metabolite conjugated with succinic acid has been identified and confirmed by synthesis. According to the reviewed literature, this is the first time that this type of conjugate is reported for human metabolism.

Full text of DOI:10.1124/dmd.114.061416

Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2014/12/02/dmd.114.061416.DC1.html
1521-009X/43/2/248–257$25.00
DRUG METABOLISM AND DISPOSITION
http://dx.doi.org/10.1124/dmd.114.061416
Drug Metab Dispos 43:248–257, February 2015
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
Mass Spectrometric Evaluation of Mephedrone In Vivo Human
Metabolism: Identification of Phase I and Phase II Metabolites,
Including a Novel Succinyl Conjugate s
Óscar J. Pozo, María Ibáñez, Juan V. Sancho, Julio Lahoz-Beneytez, Magí Farré, Esther Papaseit,
Rafael de la Torre, and Félix Hernández
Bioanalysis Research Group, Institut Hospital del Mar d’Investigacions Mèdiques, Hospital del Mar Medical Research Institute,
Barcelona, Spain (O.J.P.); Research Institute for Pesticides and Water, University Jaume I, Castellón, Spain (M.I., J.V.S., F.H.);
Human Pharmacology and Clinical Neurosciences Research Group, Institut Hospital del Mar d’Investigacions Mèdiques, Hospital
del Mar Medical Research Institute, Universitat Autònoma de Barcelona and Universitat Pompeu Fabra, Barcelona, Spain (J.L.-B.,
M.F., E.P., R.d.l.T.); and Warwick Systems Biology Centre, University of Warwick, Coventry, United Kingdom (J.L.-B.)
Received September 29, 2014; accepted December 2, 2014
ABSTRACT
In recent years, many new designer drugs have emerged, including
the group of cathinone derivatives. One frequently occurring drug is
mephedrone; although mephedrone was originally considered as
a “legal high” product, it is currently banned in most Western countries.
Despite the banning, abuse of the drug and seizures are continuously
ingested 200 mg mephedrone orally, were taken before administration
and 4 hours after substance intake. The discovery and identification
of the phase I and phase II metabolites of mephedrone were based
on ultra-high-performance liquid chromatography coupled to hybrid
quadrupole time-of-flight mass spectrometry, operating in the so-called
reported. Although the metabolism of mephedrone has been studied in MSE mode. Six phase I metabolites and four phase II metabolites
rats or in vitro using human liver microsomes, to the best of our
knowledge, no dedicated study with human volunteers has been
performed for studying the in vivo metabolism of mephedrone in
humans. Therefore, the aim of this study was to establish the actual
human metabolism of mephedrone and to compare it with other
models. For this purpose, urine samples of two healthy volunteers, who
were identified, four of them not previously reported in the literature.
The structure of four of the detected metabolites was confirmed by
synthesis of the suggested compounds. Remarkably, a mephedrone
metabolite conjugated with succinic acid has been identified and
confirmed by synthesis. According to the reviewed literature, this is the
first time that this type of conjugate is reported for human metabolism.
Introduction
been detected in numerous postmortem biologic samples of fatal cases
in which the cause of death was not specifically mephedrone (Torrance
and Cooper, 2010; Maskell et al., 2011; Schifano et al., 2012; Cosbey
et al., 2013). Abuse of mephedrone has been documented since 2007; it
was originally a “legal high” drug, but has now been banned in most
Western countries. Therefore, an intake of this drug must be monitored
in clinical and forensic toxicology and doping control. The detection of
urinary metabolites is the most suitable strategy for this purpose (Maurer
et al., 2011).
The stimulant designer drug mephedrone (M-CAT, Meow Meow) is
a derivative of cathinone, a monoamine alkaloid found in khat, the
effects of which resemble that of MDMA (3,4-methylenedioxymeth-
amphetamine; ecstasy). There are several reported fatalities in Europe
in which mephedrone intoxication appears to be the sole cause of
death (Torrance and Cooper, 2010; Lusthof et al., 2011; Maskell et al.,
2011; Adamowicz et al., 2013; Cosbey et al., 2013), and it has also
The metabolism of mephedrone has been studied mainly in rats (Meyer
et al., 2010; Martínez-Clemente et al., 2013) or in vitro using either rat
This research was supported by the Spanish Ministry of Economy and
Competitiveness [Grant Ciencias y Tecnologías Químicas (CTQ) 2012-36189], the liver hepatocytes (Khreit et al., 2013) or human liver microsomes
Ministry of Health [Grant Instituto de Salud Carlos III (ISC-III) Proyecto de Investigación
(Pedersen et al., 2013). Some of the metabolites found in these studies
have also been found in human urine samples submitted for drug
testing; however, to our knowledge, no dedicated study with human
subjects has been performed for studying the in vivo metabolism of
mephedrone in humans.
Liquid chromatography (LC) coupled to tandem mass spectrometry
(MS/MS) and/or to high-resolution mass spectrometry (MS) is one of
(PI) 11/01961], Departament d’Innovació, Universitats i Empresa (DIUE) de la
Generalitat de Catalunya [Grant 2014 Grup de recerca Reconegut de la Generalitat
(SGR) 680], and Generalitat Valenciana [Research Group of Excellence Grants
PROMETEO II/2014/023 and Institutos Superiores de Investigación Cooperativa
(ISIC) 2012/016]. This research was also supported in part by Instituto de Salud
Carlos III-Fondo Europeo de Desarrollo Regional (FEDER) [Grants Fondo de
Investigación Sanitaria (FIS) PI11/0196 and Red trastornos adictivos (RTA) RD12/
the most useful analytical tools for metabolic studies. It allows for
the direct and simultaneous detection of both phase I and phase II
metabolites. The versatility of LC combined with different analyzers
0028/0009 and Rio Hortega Fellowship ISC-III-CM13/00016].
dx.doi.org/10.1124/dmd.114.061416.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: ACN, acetonitrile; CID, collision-induced dissociation; HCOOH, formic acid; HPLC, high-performance liquid chromatography;
LC, liquid chromatography; MDMA, 3,4-methylenedioxymethamphetamine; MeOH, methanol; MS, mass spectrometry; MS/MS, tandem mass
spectrometry; m/z, mass-to-charge ratio; QTOF, hybrid quadrupole time-of-flight; UHPLC, ultra-high-performance liquid chromatography.
248

Mephedrone Metabolism in Humans
249
Calibration of the mass axis from m/z 50 to 1000 was conducted daily with
a 1:1 mixture of 0.05 M sodium hydroxide/5% (v/v) HCOOH diluted (1:25)
with water/ACN (20:80 v/v).
For automated accurate mass measurement, the lock-spray probe was used,
pumping leucine enkephalin (2 mg/l) in ACN/water (0.1% HCOOH, 50/50) at
20 ml/min through the lock-spray needle. The leucine enkephalin [M + H]+ ion
(m/z 556.2771) for positive ionization mode, and [M 2 H]2 ion (m/z 554.2615)
for negative ionization were used for recalibrating the mass axis and to ensure
a robust accurate mass measurement over time.
favored the development of a large number of analytical approaches
for the detection of metabolites (Prakash et al., 2007; Gomez et al.,
2014). Among them, ultra-high-performance liquid chromatography
(UHPLC) coupled to high-resolution MS, specifically using hybrid
quadrupole time-of-flight (QTOF) MS, is one of the most attractive for
the open detection of unknown metabolites. This configuration allows
for operating in the so-called MSE mode (i.e., two accurate mass full
spectra are acquired sequentially). The first one is acquired without
applying collision energy, providing information about the intact
molecules [commonly the (de)protonated molecule] present in the urine
sample. The second, applying a collision energy ramp, promotes
fragmentation obtaining accurate mass fragment ions useful for
metabolite identification. This approach has been successfully applied
for the detection of metabolites/transformation products of several
xenobiotics such as omeprazole (Boix et al., 2014b), cocaine and
benzoylecgonine (Bijlsma et al., 2013), and cannabis (Boix et al., 2014a).
MS data were acquired in centroid mode and processed by the MetaboLynx
XS application manager (within MassLynx version 4.1; Waters Corporation).
Data Processing
MetaboLynx XS software was used to process QTOF MS data obtained
from metabolic studies. This software compares extracted ion chromatograms
of a positive sample with a control sample for detecting, identifying, and reporting
differential ions/chromatographic peaks that would correspond, in principle, to
In this study, the human metabolism of mephedrone was evaluated metabolites. In previous studies (Boix et al., 2013, 2014a, b), MetaboLynx XS
proved to be highly useful for the investigation of both expected and unexpected
metabolites/transformation products.
using UHPLC-QTOF MS. For this purpose, urine samples collected at
predose and 4 hours after drug administration were analyzed. Potential
structures for the detected metabolites were established based on their
mass spectrometric behavior. Metabolic pathways found in humans
were compared with those obtained with other models such as rats or
microsomes.
Acquisitions were performed in centroid, in both positive and negative ion
modes. For all compounds detected by MetaboLynx, the accurate mass of
protonated/deprotonated molecules was determined on the basis of averaged
spectra obtained in the survey scan. Then, possible elemental compositions were
calculated using the MassLynx elemental composition calculator with a maximum
deviation of 2 mDa from the measured accurate mass. The maximum and
minimum parameters were restricted considering the elemental composition and
structure of mephedrone (C11H15NO) and the possibility of phase II metabolism,
such as glucuronidation, as follows: C 0–25, H 0–50, N 0–2, O 0–12, and S 0–1.
Materials and Methods
Reagents and Chemicals
Mephedrone, mephedrone-d3, and nor-mephedrone reference standards were The applied double-bond equivalent filter was set between –0.5 and 50. To
purchased from LGC GmbH (Lukenwalde, Germany) and Toronto Research calculate the elemental composition of fragment ions, parameters settings were
Chemicals (North York, Ontario, Canada). NaBH4 and succinic anhydride were restricted as a function of the calculated elemental composition of the (de)
obtained from Sigma-Aldrich (St. Louis, MO). High-performance liquid chro- protonated molecule, whereas no restrictions were applied for neutral losses. In
matography (HPLC)–grade water was obtained from deionized water passed addition, the option “even-electrons ions only” was selected for the precursor ion,
through a Milli-Q water purification system (Millipore, Bedford, MA). HPLC- and “odd- and even-electrons ions” for the fragment ions.
grade methanol (MeOH), HPLC-grade acetonitrile (ACN), sodium hydroxide,
and formic acid (HCOOH) were purchased from ScharLab (Barcelona, Spain).
Leucine enkephalin was purchased from Sigma-Aldrich.
Reduction of Reference Material: Synthesis of Reference Standards for M3
and M5
Qualitative reduction of reference material was performed based on
a procedure described elsewhere (Pozo et al., 2012), in which the carbonyl
Instrumentation
An Acquity ultra-performance LC system (Waters, Milford, MA) was interfaced group is converted in an alcohol moiety. Briefly, 100 ml of reference material
to a QTOF mass spectrometer (QTOF Xevo G2; Waters Micromass, Manchester, (both mephedrone and nor-mephedrone) at 100 mg/ml was evaporated to
UK) using an orthogonal Z-spray electrospray interface. The LC separation was
performed using an Acquity ultra-performance LC BEH C18 1.7-mm particle size maintained under agitation for 3 hours. After evaporation of the solvent, the
analytical column of 100 Â 2.1 mm (from Waters) at a flow rate of 0.3 ml/min.
residue was dissolved in 1 ml of mobile phase. The presence of the reduced
The mobile phases used were H2O (A) and MeOH (B), both with 0.01% (v/v) metabolites was confirmed by UHPLC-QTOF MS analysis using the above-
dryness. After addition of 2 ml methanol and 100 mg NaBH4, the mixture was
HCOOH. The proportion of MeOH was linearly increased as follows: 0 minute,
10%; 14 minutes, 90%; 16 minutes, 90%; 16.01 minutes, 10%; and 18 minutes,
10%. The injection volume was 20 ml. Nitrogen (Praxair, Valencia, Spain) was
used as both the drying and nebulizing gas. The gas flow rate was set at 1000 l/h.
The resolution of the time-of-flight mass spectrometer was approximately 20,000
at full width half maximum at a mass-to-charge ratio (m/z) of 556. MS data were
acquired over a m/z range of 50–1000 in a scan time of 0.3 seconds. The
microchannel plate (MCP) detector potential was set to 3700 V. Capillary voltages
of 0.7 kV and 22.0 kV were used in positive and negative ionization modes,
respectively. A cone voltage of 15 V was applied. The collision gas was argon
(99.995%; Praxair). The interface temperature was set to 600ꢀC and the source
temperature to 130ꢀC. The column and autosampler temperatures were set to 40ꢀC
and 5ꢀC, respectively.
described experimental conditions.
Synthesis of Nor-Mephedrone Succinate (M4)
Nor-mephedrone conjugated with succinic acid was qualitatively synthe-
sized by mixing 100 ml nor-mephedrone at 100 mg/ml with 100 mg succinic
anhydride in acetone. The mixture was heated at 60ꢀC for 3 hours. After
evaporation of the solvent, the residue was dissolved in mobile phase. Residues
of insoluble salts were removed by centrifugation. The presence of the
succinate metabolite was confirmed by UHPLC-QTOF MS analysis using the
above-described experimental conditions.
Urine Samples
For MSE experiments, two acquisition functions with different collision energies
were created: the low-energy function with a collision energy of 4 eV, and the
high-energy function with a collision energy ramp ranging from 15 to 30 eV.
Urine samples were obtained predose and after 4 hours from two volunteers that
were administered with a single oral dose of 200 mg mephedrone in the Institut
Hospital del Mar d’Investigacions Mèdiques Clinical Research Unit. Subjects were
MS/MS experiments were also performed, using a cone voltage of 15 V and two Caucasian healthy male recreational users of psychostimulants and halluci-
collision energies of 10, 15, and 20 eV. In those cases in which a single nogens (ages 29 and 36 years). Both participants were extensive-intermediate
abundant product ion was obtained, a MS3 experiment was performed by CYP2D6 metabolizers. They were free of any medicine or drugs of abuse. A rapid
selecting the product ion generated in-source at high cone energy (30 V) urine test for main drugs of abuse was negative before administration. Subjects
and acquiring the product ion scan of this in-source fragment.
signed an informed consent and the protocol was authorized by the local ethics

250
Pozo et al.
committee (Clinical Research Ethical Committee–Parc de Salut Mar, reference no.
IMIMFTCL/MEF/1, 2013/5045).
unconjugated metabolite. The results of these experiments are shown
in Table 1.
Sample Treatment
Mass Spectrometric Results for Mephedrone Metabolites
One milliliter of the urine sample was diluted 2-fold with Milli-Q water and
centrifuged at 10,000 rpm for 7 minutes. After that, 20 ml was directly injected
in the LC-QTOF MS system.
Metabolite M1. The molecular formula obtained for metabolite M1
(C10H13NO) suggested the loss of a methyl group from mephedrone.
Because N-demethylation is a common metabolic pathway of
N-methyl stimulants, N-demethylmephedrone was postulated as a
feasible structure for M1.
As expected, the product ion spectrum followed behavior similar to
the observed for mephedrone. Thus, the expected neutral loss of
ammonia was substantially less abundant than the losses of water and
CH3• (Table 1). This fact can be explained by the same rearrangement
postulated for mephedrone (Supplemental Fig. 1).
Results
Mass Spectrometric Behavior of Mephedrone
Similar to other nitrogen-containing stimulants, mephedrone (C11H15NO)
is easily ionized in positive ionization mode producing an abundant
[M + H]+. The ions formed in the collision-induced dissociation (CID)
of mephedrone are shown in Table 1. Some ions resulting from neutral
losses of water and a methyl group are common for similar molecules,
such as amphetamine and ephedrine derivatives (Deventer et al., 2009;
Bijlsma et al., 2011). However, the observed MS/MS behavior of
mephedrone was to some extent unexpected. Nitrogen-containing
stimulants usually lose the amine group (normally as ammonia or
methylamine) in one of the first fragmentation steps (Deventer et al.,
2009; Bijlsma et al., 2011). Contrary to this behavior, the neutral loss
of methylamine is poor (2%; see Table 1) for mephedrone. In fact,
the loss of the nitrogen atom seems to be unfavored because all of
the ions with abundances higher than 20% still preserve the nitrogen
The structure of M1 was confirmed by comparison with the reference
material (Fig. 2, bottom).
Metabolite M2. The molecular formula of M2 (C10H11NO3)
indicated a double oxidation from M1. In addition, M2 was also detected
in negative ionization mode (Table 1), suggesting the presence of an
acidic moiety. Therefore, the oxidation of one of the methyl groups of
M1 to carboxylic acid was the most feasible mechanism to produce M2.
The product ion spectrum provided information about the location of the
carboxylic function. The product ion spectrum of M2 was dominated by
the ion at m/z 119.0497 (Table 1). This ion can be obtained after the neutral
loss of glycine (Supplemental Fig. 2), suggesting a proximity between the
amine and the acidic groups. In addition, the formula of this ion (C8H7O)
implied that the aromatic zone of the mephedrone remains intact.
This fact was confirmed by the fragmentation of the ion C8H7O
generated in-source. This fragmentation followed the expected behavior
for a CH3PhCO+ ion. It was dominated by the tropylium ion (m/z
91.0552) and its subsequent fragmentation (Supplemental Fig. 2). In
addition, an apparent neutral loss of 10 Da was observed. This fact can
be explained by the addition of water in the collision cell after the CO
loss as it was reported for similar ions (Beuck et al., 2009).
Based on this information, N-demethylmephedrone-3-carboxylic
acid was selected as the potential structure for M2.
•
(Table 1). Two losses of radical CH3 were observed without losing
the amine. This behavior can be considered as exceptional because
the formation of odd electron ions by losses of radicals is uncommon
in CID (Thurman et al., 2007).
This unusual behavior may be explained by an in-source rearrange-
ment previous to the fragmentation (Fig. 1, top). This rearrangement
would form an indole ring by reaction between the nitrogen atom and the
C2, with the help of the carbonyl function at C1. The stability provided
by the indole would explain the unusual behavior of mephedrone
including the abundant loss of water (coming from the ketone) and the
•
subsequent losses of CH3 . The analysis of mephedrone-d3 confirmed
that both methyl groups could be lost, although the loss of the C3 methyl
group seems to be favored because the ion at m/z 148 is more abundant
than the ion at m/z 145 (Fig. 1, bottom).
Metabolite M3. The molecular formula obtained for M3 (C11H17NO)
indicated a reduction step from mephedrone.
The product ion spectrum exhibited an abundant neutral loss of
water and subsequent losses of CH3• and methylamine (Table 1). This
Detection of Mephedrone Metabolites
The strategy applied in this study (dilution of the sample, acquisition behavior is similar to the one observed for ephedrine derivatives that
in UHPLC-QTOF MS, and processing by MetaboLynx software) to have a hydroxyl group in C1. Therefore, the reduction of the ketone
urine samples collected before and after administration of 200 mg moiety would hamper the rearrangement postulated for mephedrone,
mephedrone allowed for the detection of the parent compound and 10 decreasing the number of nitrogen-containing product ions (Supple-
metabolites (Table 1).
mental Fig. 3).
The mass accuracy obtained provided only one feasible formula for
The structure of this metabolite as 1-dihydromephedrone was con-
each metabolite at the selected narrow-mass window (2 mDa). Among firmed by comparison with synthesized material (Fig. 2, middle).
the 10 metabolites found, 4 were detected only in the positive ionization
Metabolite M4. Metabolite M4 exhibited a molecular formula
mode, similar to the behavior observed for mephedrone. Six of the containing three carbon atoms more than mephedrone (C14H17NO4).
metabolites were also detected in negative ionization mode (Table 1), This fact suggested that M4 was conjugated by phase II metabolism. A
suggesting the presence of an acidic moiety.
diacetyl metabolite with the same formula was reported in the in vitro
The product ion scan of each detected metabolite was acquired and metabolism of mephedrone (Khreit et al., 2013). However, M4 showed
the results are shown in Table 1. Based on the comparison between these ionization in both positive and negative modes (Table 1), indicating the
results and the CID behavior of mephedrone (see above), a putative presence of an acidic moiety in M4. This result is in disagreement with
structure for each metabolite was established (described below).
In those cases in which a phase II metabolite was predicted (M4, M8,
the diacetyl metabolite.
The product ion spectrum of M4 was one of the most complex among
M9, and M10) or a single abundant product ion was obtained (M2 the detected metabolites (Fig. 3A). It showed that the molecule could be
and M7), an MS3 experiment was performed by selecting the product divided in two parts: one fragment at m/z 164 (C10H14NO) and the other
ion generated in-source at high cone voltage and acquiring the prod- at m/z 101 (C4H5O3). The fragment C10H14NO has the same molecular
uct ion scan of this in-source fragment. This allowed us to garner more formula as M1 (i.e., nor-mephedrone). Therefore, the fragment at m/z
knowledge or to obtain additional relevant information about the 101 provided information about the phase II metabolite. The molecular

Mephedrone Metabolism in Humans
251
TABLE 1
Mass spectrometric results for the detected metabolites
Metabolite
Rt
Ionization Mode
ESI+
m/z
Error
Formula
Precursor Ion
Product Ion
Abundance (%)
Error
Formula
min
min
%
mDa
Mephedrone
4.16
178.1231
20.1
C11H16NO
178
160.1125
147.0815
145.0889
144.0813
130.0663
119.0862
117.0704
91.0548
45
2
100
22
2
15
2
4
20.1
0.5
20.2
0
0.6
0.1
C11H14
N
O
N
C
C
10H11
10H11
C
10H10NO
C9H8N
C9H11
C9H9
0
0
C7H7
79.0546
2
8
20.2
20.7
0.2
0.2
0.5
0.1
0.3
0.2
C6H7
C10H11
C10H12
C9H9N
C9H8N
C9H11
C9H9
M1
M2
3.87
3.84
ESI+
ESI+
164.1078
194.0823
0.3
0.6
C10H14NO
164
147.0803
146.0972
131.0737
130.0662
119.0862
117.0707
91.0550
O
N
50
100
20
22
5
8
C7H7
79.0547
2
20.1
21.5
0
0.1
20.1
0.4
C6H7
C10H12NO3
194
119
138.0666
119.0497
109.0654
91.0547
109.0657
91.0546
12
100
5
20
10
100
5
C8H10O2
C8H7O
C7H9O
C7H7
C7H9O
C7H7
20.2
0
77.0391
C6H5
65.0387
8
100
8
32
100
70
12
32
8
7
18
5
8
8
20.4
0.1
C5H5
ESI2
192.0663
180.1387
0.2
C10H10NO3
C11H18NO
192
180
148.0763
146.0603
91.0546
162.1276
147.1028
146.0971
131.0861
129.0706
105.0706
91.0547
C9H10NO
C9H8NO
C7H7
C11H16
10H13
10H12
C10H11
C10H9
C8H9
C7H7
C4H8N
C14H16NO3
C14H14NO2
C12H12NO
C10H14NO
10H11
20.3
20.2
20.7
22.0
0.1
0
0.2
0.2
20.1
20.2
20.2
20.1
0.4
0.5
0.5
0.1
21.0
0
M3
M4
3.92
7.38
ESI+
20.1
N
N
N
C
C
70.0655
ESI+
264.1234
20.2
C
14H18NO4
264
246.1115
228.1024
186.0923
164.1084
147.0815
146.0971
131.0725
126.0555
119.0860
101.0238
98.0617
9
18
40
100
10
62
58
22
7
C
O
N
C10H12
C9H9O
C6H8NO2
C9H11
C4H5O3
C5H8NO
20.1
20.1
1.1
164
262
146.0971
131.0725
119.0859
117.0705
91.0548
48
100
28
5
8
2
78
42
100
18
18
38
25
72
100
28
12
5
0.1
C10H12N
C9H9O
C9H11
C9H9
C7H7
21.0
20.2
0.1
0
0.6
20.2
22.1
20.4
1.3
21.4
20.3
2.9
20.4
0
0.1
20.3
20.1
0
21.2
21.8
20.9
20.6
20.2
67.0554
C5H7
ESI2
ESI+
ESI+
262.1085
166.1231
194.1178
0.6
20.1
0.3
C14H16NO4
244.0972
226.0847
200.1071
162.0932
133.0639
99.0079
C14H14NO3
C14H12NO2
C13H14NO
C
10H12NO
C9H9O
C4H3O3
C4H4NO2
98.0271
M5
M6
3.84
2.61
C10H16NO
166
194
148.1122
131.0861
116.0627
115.0545
105.0703
91.0548
C10H14
N
C
10H11
C9H8
C9H7
C8H9
C7H7
C3H6N
C11H14NO
60
2
2
100
100
8
56.0488
C
11H16NO2
176.1057
158.0961
146.0962
133.0651
C
C
11H12
10H12
N
N
C9H9O
(continued)

252
Pozo et al.
TABLE 1—Continued
Metabolite
Rt
Ionization Mode
ESI+
m/z
Error
Formula
Precursor Ion
Product Ion
Abundance (%)
Error
Formula
131.0728
105.0696
190.0879
172.0762
146.0968
144.0813
131.0735
130.0656
105.0702
162.0914
160.0766
147.0696
84.0438
209.1169
194.1176
191.1075
178.1234
160.1121
119.0497
161.0818
148.1096
119.0502
109.0656
91.0554
43
12
5
35
81
18
100
8
20
100
62
20
8
12
100
22
8
20.7
20.8
1.1
C9H9N
C8H9
C11H12NO2
C11H10NO
M7
1.19
208.0973
20.1
C11H14NO3
208
0
20.2
0
0
20.1
20.2
20.5
0.4
C
C
10H12
10H10
N
N
C9H9N
C9H8N
C8H9
C10H12NO
10H10NO
C9H9NO
C4H6NO
C12H17O3
C11H16NO2
C12H15O2
C11H16NO
C11H14N
C8H7O
10H11NO
C7H16O3
C8H7O
C7H9O
C7H7
C6H7
C6H9O7
C6H5O5
C5H7O4
C5H5O3
C3H3O4
C2HO3
C11H14NO3
C11H12NO2
C11H10NO
ESI2
206.0812
370.1509
20.5
C11H12NO3
206
162
C
1.2
20.1
20.9
20.5
0.3
0.2
20.5
0
0.7
20.3
0.5
0.3
0.6
20.3
21.3
0
0.7
0.4
20.9
20.9
0.8
20.6
0.1
20.2
21.5
0.1
M8
6.62
ESI+
0.7
C
17H24NO8
370
194
368
32
68
10
8
100
8
C
35
5
79.0545
ESI2
368.1358
384.1302
1.1
0.7
C17H22NO8
193.0335
157.0137
131.0350
113.0239
103.0022
72.9917
208.0982
190.0862
172.0763
146.0968
190.0853
172.0763
146.0972
131.0737
105.0707
206.0812
162.0915
113.0234
85.0280
162.0923
160.0762
77.0386
180.1028
162.0919
119.0478
149.0974
131.0864
107.0867
91.0543
62
18
38
100
40
32
100
22
58
32
5
25
82
100
22
100
58
72
22
100
8
M9
1.27
ESI+
C17H22NO9
384
208
C
10H12
11H12NO2
C11H10NO
10H12
N
C
0.2
0.2
0.3
C
N
C9H9N
C8H9
C11H12NO3
C10H12NO
C5H5O3
ESI2
ESI+
ESI2
382.1128
356.1346
354.1185
21.0
C17H20NO9
C16H22NO8
C16H20NO8
382
20.5
20.4
20.5
21.0
0.4
20.9
20.5
0.3
C4H5O2
C10H12NO
206
356
180
C
10H10NO
C6H5
C10H14NO2
C10H12NO
C8H7O
5
M10
5.71
0.1
100
72
45
22
100
18
50
38
42
18
0
21.9
0.8
0.3
0.6
20.5
1.1
C
10H13
O
C10H11
C8H11
C7H7
C9H7NO4
C5H5O3
C4H5O2
20.4
354
193.0364
113.0237
85.0303
20.2
1.3
ESI, electrospray ionization; Rt, Retention time.
formula was in agreement with a succinyl conjugate. The product ion rearrangement observed in mephedrone. In agreement with this, the
spectra could be explained by this conjugation as shown in Fig. 3C. product ion spectrum of M5 was dominated by subsequent neutral losses
The structure of M4 as N-succinyl nor-mephedrone was confirmed of water and ammonia (Supplemental Fig. 4).
by comparison with synthetic material as shown in Fig. 3B.
The proposed structure was confirmed by the reduction of nor-
Metabolite M5. Metabolite M5 showed only positive ionization with mephedrone, as can be seen in Fig. 2 (top).
a molecular formula of C10H15NO. This formula suggested a demethyl-
Metabolite M6. The molecular formula obtained for M6 (C11H15NO2)
ation followed by a reduction of the keto function. Therefore, indicated a hydroxylation from mephedrone. Since mephedrone contains
1-dihydro-nor-mephedrone was selected as the most feasible candidate several potential hydroxylation sites, the product ion spectrum was used
for M5.
This hypothesis was supported by the product ion spectrum. Similar
to propose the most feasible one.
The product ion spectrum showed the two expected losses of water:
to M3, the absence of the ketone functionality seems to hamper the the one coming from the ketone observed in most of the mephedrone

Mephedrone Metabolism in Humans
253
Fig. 1. Proposed fragmentation pathway for mephedrone reference standard (top), product ion spectrum at 20 eV for mephedrone (middle), and product ion spectrum at
20 eV for mephedrone-d3 (bottom).
metabolites and the other coming from the incorporated hydroxyl stable rings, such as steroids (Pozo et al., 2008). Therefore,
group. In addition, an abundant neutral loss of formaldehyde was also 49-hydroxymethyl-mephedrone was proposed for M6 in agreement
observed (Table 1; Supplemental Fig. 5). This neutral loss of 30 Da with previously published studies (Meyer et al., 2010; Khreit et al.,
has been reported for hydroxymethyl metabolites in compounds with 2013; Martínez-Clemente et al., 2013; Pedersen et al., 2013).

254
Pozo et al.
Fig. 2. MS/MS spectra at 20 eV for metabolites found in urine samples (A) and synthesized materials/reference standards (B).
Metabolite M7. The molecular formula of M7 (C11H13NO3) and corresponding to the unconjugated metabolite) showed an abundant
suggested a double oxidation from mephedrone. Similar to M2, the ion at m/z 119.0497 (Table 1) corresponding to CH3-Ph-CO+. As
oxidation of a methyl group to carboxylic acid seems to be the most commented for other metabolites, this ion is indicative of unaltered
feasible mechanism to obtain M7. This fact was also supported by the benzylic zone in M8. Therefore, C3 seems to be the most suitable
negative ionization observed for M7 (Table 1).
hydroxylation site. Similarly to M2 in which a carboxylic acid was
In contrast with M2, the product ion spectrum of M7 did not show proposed in C3, M8 also showed the ions corresponding to the loss
the ion C8H7O (m/z 119.0500) indicative of an intact benzylic area. of CO and the subsequent gain of a water molecule (Supplemental
•
The spectrum of M7 was dominated by losses of H2O, CO2, and CH3 . Fig. 7), supporting the assignation. Therefore, a C3 hydroxylation previous
These losses could be explained after the rearrangement described for to glucuronidation is proposed as the most feasible metabolic pathway
mephedrone if the structure of M7 is 49-carboxymephedrone (Supple- to obtain M8.
mental Fig. 6) as previously reported (Khreit et al., 2013; Pedersen
et al., 2013; Martínez-Clemente et al., 2013).
Metabolite M9. The molecular formula for M9 (C17H21NO9) also
suggested a glucuronide conjugate. In that case, the phase I metabolite
Metabolite M8. M8 showed a molecular formula of C17H23NO8, seemed to be a double oxidized metabolite.
which implied an increase of C6H8O6 from hydroxylmephedrone. This
The product ion spectrum of M9 after the loss of glucuronide
increase is associated with a glucuronide moiety. In addition, M8 was moiety was identical to the one observed for M7 (Table 1;
detected in both positive and negative ionization modes (Table 1). Supplemental Fig. 8). Therefore, the glucuronide conjugate of
Therefore, hydroxylmephedrone-3-O-glucuronide was proposed as a fea- 49-carboxymephedrone was proposed as the structure of M9. Based
sible structure for M8.
on the structure of M7, N-glucuronidation seems to be the most
The product ion spectrum confirmed the presence of the glucuronide feasible metabolic pathway to obtain M9 (49-carboxymephedrone-N-
moiety by the typical loss of 176 Da in positive ionization mode and the glucuronide).
occurrence of the ion at m/z 113 in negative ionization mode associated
Metabolite M10. Similarly to M8 and M9, the molecular formula
with the glucuronide functionality (Fabregat et al., 2013). In addition, of M10 (C16H20NO8) indicated a glucuronidation. In that case, the
the product ion spectrum helped in the assignation of the hydroxylation molecular formula of the phase I metabolite produced after the loss of
site (Supplemental Fig. 7). The product ion spectrum of the in-source the glucuronide moiety (C10H13NO2) corresponded to a hydroxylated
fragment at m/z 194 (produced after the neutral loss of the glucuronide metabolite of nor-mephedrone.

Mephedrone Metabolism in Humans
255
Fig. 3. MS/MS spectra at 20 eV in positive ionization mode (top) and negative ionization mode (bottom) for M4 (N-succinyl nor-mephedrone) in urine (A) and synthesized
reference standard (B). (C) Proposed fragmentation pathway for this metabolite.
The product ion spectrum of M10 exhibited an abundant ion at m/z Fig. 9). Following the considerations described for these metabolites,
119.0478 (C8H7O). As described for other metabolites such as M2 hydroxylation in C3 of nor-mephedrone and subsequent glucuronidation
and M8, this ion is indicative of an intact aromatic area (Supplemental was proposed as a potential metabolic pathway to obtain M10 (hydroxyl

256
Pozo et al.
nor-mephedrone-3-O-glucuronide). The C3-hydroxylated metabolite pre- of the nitrogen atom. Therefore, discerning between both C9H11 and
cursor of M10 might also be proposed as an intermediate metabolite in C8H7O formulae for the ion at m/z 119 was found to be compulsory
the generation of M2 (Fig. 4).
for structural elucidation.
In summary, six phase I and four phase II mephedrone metabolites
were detected and elucidated in human urine. Phase I reactions included
N-demethylation, reduction of the keto function, hydroxylations in C3
Discussion
The study of mephedrone metabolism by combination of UHPLC- and in the benzylic carbon, and oxidation of C3 and the benzylic methyl
QTOF MS and MetaboLynx after dilution of the sample allowed for the to carboxylic acid (Fig. 4). Our data confirmed the presence of some
detection of mephedrone and 10 metabolites in human urine. The compounds that were previously described as mephedrone metabolites
metabolic pathway proposed based on our results is summarized in Fig. 4. in humans or other species (e.g., M1, M3, M5, M6, M7, and M9)
The accurate mass measurements played a critical role in properly (Meyer et al., 2010; Khreit et al., 2013; Martínez-Clemente et al., 2013;
interpreting the fragmentation of mephedrone and proposing suitable Pedersen et al., 2013). However, it is remarkable that structures for M2,
structures for detected metabolites. For example, mephedrone and M4, M8, and M10 are reported for the first time in this study.
most of its metabolites show a product ion with nominal m/z 119. In
Regarding phase II metabolism, three metabolites conjugated with
a previous report, this ion was assigned to CH3PhCO+, suggesting glucuronic acid (M8, M9, and M10) were found. More surprising was
a cleavage of the C1-C2 bond of the molecule (Martínez-Clemente the detection of a conjugate with succinic acid (M4). To the best of our
et al., 2013). However, accurate mass measurements revealed that it knowledge, this is the first report of such a conjugate in human urine.
corresponds to a formula of C9H11, indicating that the expected Succinylation in drug development falls into the prodrug category.
cleavage is not produced. By contrast, several metabolites produced Succinic acid derivatives are attached to target drugs to produce more
the ion CH3PhCO+, which was confirmed by the subsequent loss of desirable physical and biologic properties, with the goal that the
CO and the ion at m/z 109 corresponding to [CH3PhCO – CO + H2O]+. The resulting conjugates are degraded by cellular enzymes to release the
presence of this ion would imply a structural change in the surroundings target drugs in vivo. In the context of physiologic compounds, biogenic
Fig. 4. Metabolic pathway proposed for mephedrone in humans.

Mephedrone Metabolism in Humans
257
Bijlsma L, Sancho JV, Hernández F, and Niessen WMA (2011) Fragmentation pathways of drugs
of abuse and their metabolites based on QTOF MS/MS and MS(E) accurate-mass spectra.
J Mass Spectrom 46:865–875.
amines such as octopamine, dopamine, and serotonin are typically
deactivated by acetylation in insects, nematodes, and other invertebrates.
Succinylation, in addition to acetylation, was recently reported for
octopamine and tyramine ascarosides in nematodes, raising the question
of to what extent this metabolic reaction may represent a general
pathway of biogenic amine metabolism (Artyukhin et al., 2013). In the
context of our study, succinic acid was conjugated to nor-mephedrone.
The hydrolysis of the conjugate would release nor-mephedrone,
a metabolite which pharmacology activity is unknown but expected to
be lower than the corresponding to mephedrone.
The structure of four of the detected metabolites (M1, M3, M4, and
M5) was confirmed by comparison with synthesized material. All four
were correctly assigned based on their mass spectrometric behavior.
Synthesis of the remaining metabolites would be required to ultimately
confirm their structure.
In vitro studies suggest that mephedrone metabolic disposition is
regulated by the highly polymorphic isoenzyme of CYP2D6 (Pedersen
et al., 2013). The quantification of metabolites identified in this report is
of importance since it is unknown i) the in vivo relevance of CYP2D6
polymorphism in mephedrone disposition and ii) the metabolic
pathways regulated by this isoenzyme to interpret metabolic profiles
in the context of forensic toxicology.
In the near future, research is needed to evaluate the behavior of
unchanged mephedrone and the 10 reported metabolites in urine
samples collected from a larger population, ideally after administration
of different doses, to determine which should be the most appropriate
biomarkers when investigating drug use.
Boix C, Ibáñez M, Bijlsma L, Sancho JV, and Hernández F (2014a) Investigation of cannabis
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Boix C, Ibáñez M, Sancho JV, Niessen WMA, and Hernández F (2013) Investigating the pres-
ence of omeprazole in waters by liquid chromatography coupled to low and high resolution
mass spectrometry: degradation experiments. J Mass Spectrom 48:1091–1100.
Boix C, Ibáñez M, Zamora T, Sancho JV, Niessen WMA, and Hernández F (2014b) Identification
of new omeprazole metabolites in wastewaters and surface waters. Sci Total Environ 468-469:
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Deventer K, Pozo OJ, Van Eenoo P, and Delbeke FT (2009) Development and validation of an
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Fabregat A, Pozo OJ, Marcos J, Segura J, and Ventura R (2013) Use of LC-MS/MS for the
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5005–5014.
Khreit OI, Grant MH, Zhang T, Henderson C, Watson DG, and Sutcliffe OB (2013) Elucidation
of the Phase I and Phase II metabolic pathways of (6)-49-methylmethcathinone (4-MMC) and
(6)-49-(trifluoromethyl)methcathinone (4-TFMMC) in rat liver hepatocytes using LC-MS and
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Mephedrone pharmacokinetics after intravenous and oral administration in rats: relation to
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Maskell PD, De Paoli G, Seneviratne C, and Pounder DJ (2011) Mephedrone (4-methylmethcathinone)-
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Maurer HH, Pfleger K, and Weber AA (2011) Mass Spectral and GC Data of Drugs, Poisons,
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Meyer MR, Wilhelm J, Peters FT, and Maurer HH (2010) Beta-keto amphetamines: studies on the
metabolism of the designer drug mephedrone and toxicological detection of mephedrone,
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mephedrone and analysis of forensic cases. Drug Test Anal 5:430–438.
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Collision-induced dissociation of 3-keto anabolic steroids and related compounds after elec-
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Authorship Contributions
Participated in research design: Pozo, Sancho, Farré, de la Torre, Hernandez.
Conducted experiments: Pozo, Ibañez, Lahoz-Beneytez, Farré, Papaseit, de
la Torre.
Performed data analysis: Pozo, Ibañez, Sancho.
Wrote or contributed to the writing of the manuscript: Pozo, Ibañez, de la
Torre, Hernandez.
Prakash C, Shaffer CL, and Nedderman A (2007) Analytical strategies for identifying drug
metabolites. Mass Spectrom Rev 26:340–369.
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Address correspondence to: María Ibáñez, Research Institute for Pesticides and
Water, University Jaume I, Avda. Sos Baynat, E-12071 Castellón, Spain. E-mail:
ibanezm@uji.es