MASS SPECTROMETRIC EVALUATION OF …dmd.aspetjournals.org/content/dmd/early/2014/12/02/dmd.114.061416...dmd#61416 1 mass spectrometric evaluation of mephedrone in vivo human metabolism:

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MASS SPECTROMETRIC EVALUATION OF MEPHEDRONE IN VIVO

HUMAN METABOLISM: IDENTIFICATION OF PHASE I AND PHASE II

METABOLITES INCLUDING A NOVEL SUCCINYL CONJUGATE.

Óscar J. Pozo, María Ibáñez, Juan V. Sancho, Julio Lahoz-Beneytez, Magí Farré, Esther

Papaseit, Rafael de la Torre, Félix Hernández

Affiliations:

Bioanalysis Research Group, IMIM, Hospital del Mar Medical Research Institute,

Doctor Aiguader 88, 08003 Barcelona, Spain: OJP

Research Institute for Pesticides and Water, University Jaume I, Avda. Sos Baynat, E-

12071 Castellón, Spain: MI, JVS and FH

Human Pharmacology and Clinical Neurosciences Research Group, IMIM-Hospital del

Mar Medical Research Institute, Universitat Autònoma de Barcelona and Universitat

Pompeu Fabra, Barcelona, Spain: JLB, MF, EP and RT

Warwick Systems Biology Centre, University of Warwick, Coventry, United Kingdom:

JLB

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on December 2, 2014 as DOI: 10.1124/dmd.114.061416

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RUNNING TITLE:

Mephedrone metabolism in humans

Corresponding author:

María Ibáñez

Avda. Sos Baynat,

E-12071 Castellón,

Spain.

[email protected],

Tel +34 964 387339,

Fax +34 964 387368

Number of text pages: 19

Number of Tables: 1

Number of Figures: 4

Number of references: 25

Number of words in the Abstract: 247

Number of words in the introduction: 504

Number of words in the discussion: 664


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List of abbreviations:

MDMA: 3,4-methylenedioxy-methamphetamine

LC-MS/MS: Liquid chromatography coupled to tandem mass spectrometry

UHPLC: Ultra-high performance liquid chromatography

QTOF MS: Hybrid quadrupole-time of flight mass spectrometry

FWHM: Full width half maximum

ACN: Acetonitrile

CID: Collision-induced dissociation

MeOH: methanol

HCOOH: formic acid

NaOH: sodium hydroxide


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ABSTRACT

In recent years, many new designer drugs have emerged, including the group of

cathinone derivatives. One frequently occurring is mephedrone, that although was

originally considered as “legal high” product, is currently banned in most Western

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. So, 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 of mephedrone by 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 was based on ultra-

performance liquid chromatography coupled to hybrid quadrupole-time of flight mass

spectrometry (UHPLC-QTOF MS), operating in the so-called MSE 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 has been

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.


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INTRODUCTION

The stimulant designer drug mephedrone (M-CAT, Meow Meow) is a derivative of

cathinone, a monoamine alkaloid found in khat, the effects of which resembles that of

3,4-methylenedioxy methamphetamine (MDMA, ecstasy). There are several reported

fatalities in Europe in which mephedrone intoxication appears to be the sole cause of

death (Torrance at al. 2010, Maskell et al. 2011, Lusthof et al. 2011, Adamowicz et al.

2013, Cosbey et al. 2013), and it has also been detected in numerous post-mortem

biological samples of fatal cases in which the cause of death was not specifically

mephedrone (Torrance at al. 2010, Maskell et al. 2011, Schifano et al. 2013, 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 metabolism of mephedrone has been studied mainly in rats (Meyer et al., 2010,

Martinez-Clemente et al., 2013) or in vitro using either rat liver hepatocytes (Khreit et

al., 2013) or human liver microsomes (Pedersen et al., 2013). Some of the metabolites

found in these studies have also been found in human urine samples submitted for drug

testing, but no dedicated study with human subjects has been performed for studying the

in vivo metabolism of mephedrone in humans.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and/or to

high resolution (HR) MS is one of 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 liquid chromatography combined with different analyzers


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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 coupled to HR MS, specifically using hybrid

quadrupole-time of flight mass spectrometry (UHPLC-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., 2013a), cocaine and benzoylecgonine (Bijlsma et al., 2013) or cannabis (Boix et al.,

2014).

In this study, the human metabolism of mephedrone was evaluated using UHPLC-

QTOF MS. For this purpose, urine samples collected at pre-dose 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

human were compared with those obtained with other models like rats or microsomes.


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MATERIALS AND METHODS

Reagents and chemicals

Mephedrone, mephedrone-d3 and nor-mephedrone reference standards were purchased

from LGC-GmbH (Lukenwalde, Germany) and TRC (North York, Canada). NaBH4 and

succinic anhydride were obtained from Sigma Aldrich (St. Louis, MO, USA). HPLC-

grade water was obtained from deionized water passed through a Milli-Q water

purification system (Millipore, Bedford, MA, USA). HPLC-grade methanol (MeOH),

HPLC-grade acetonitrile (ACN), sodium hydroxide (NaOH) and formic acid (HCOOH)

were purchased from ScharLab (Barcelona, Spain). Leucine enkephalin was purchased

from Sigma Aldrich (St. Louis, MO, USA).

Instrumentation

An Acquity ultra-performance liquid chromatography (UPLC) system (Waters, Milford,

MA, USA) was interfaced to a QTOF mass spectrometer (QTOF Xevo G2, Waters

Micromass, Manchester, UK) using an orthogonal Z-spray electrospray interface. The

LC separation was performed using Acquity UPLC BEH C18 1.7 µm particle size

analytical column of 100 x 2.1 mm (from Waters) at a flow rate of 0.3 mL/min. The

mobile phases used were A H2O and B MeOH, both with 0.01% (v/v) HCOOH. The

proportion of MeOH was linearly increased as follows: 0 min, 10%; 14 min, 90%; 16

min, 90%; 16.01 min, 10% and 18 min, 10%. The injection volume was 20 μL.

Nitrogen (Praxair, Valencia, Spain) was used as both drying and nebulizing gas. The

gas flow rate was set at 1000 L/h. The resolution of the TOF mass spectrometer was

approximately 20,000 at full width half maximum (FWHM) at m/z 556. MS data were

acquired over a m/z range of 50–1000 in a scan time of 0.3 s. The MCP detector


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potential was set to 3700 V. Capillary voltages of 0.7 kV and -2.0 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 600 ºC and the source temperature to 130 ºC. The column and autosampler

temperatures were set to 40 ºC and 5 ºC, respectively.

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.

MS/MS experiments were also performed, using a cone voltage of 15 V and collision

energies of 10, 15 and 20 eV. In those cases in which a single abundant product ion was

obtained, a MS3 experiment was performed by selecting the product ion generated in-

source at high cone energy (30 V) and acquiring the product ion scan of this in-source

fragment.

Calibration of the mass-axis from m/z 50 to 1000 was conducted daily with a 1:1

mixture of 0.05M NaOH/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 µL/min

through the lock-spray needle. The leucine enkephalin [M+H]+ ion (m/z 556.2771) for

positive ionization mode, and [M-H]- 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.

MS data were acquired in centroid mode and processed by the MetaboLynx XS

application manager (within MassLynx v 4.1; Waters Corporation).


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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 which would correspond, in principle, to metabolites. In

previous studies (Boix et al., 2013a; Boix et al., 2013b;Boix et al., 2014), MetaboLynx

XS proved to be highly useful for the investigation of both expected and unexpected

metabolites/transformation products.

Acquisitions were performed in centroid, in both positive and negative ion mode. 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. The applied double-bond equivalent filter was set between –0.5 and 50.

To calculate the elemental composition of fragment ions, parameters settings were

restricted as a function of the calculated elemental composition of the (de)protonated

molecule, while for neutral losses no restrictions were applied. Additionally, the option

“even-electrons ions only” was selected for the precursor ion, and “odd- and even-

electrons ions” for the fragment ions.


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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), where the carbonyl group is converted in an

alcohol moiety. Briefly, 100 µL of reference material (both mephedrone and nor-

mephedrone) at 100 µg/mL were evaporated to dryness. After addition of 2 mL of

methanol and 100 mg of NaBH4, the mixture was maintained under agitation for 3 hours.

After evaporation of the solvent, the residue was dissolved in 1 mL of mobile phase.

The presence of the reduced metabolites was confirmed by UHPLC-QTOFMS analysis

using the experimental conditions described above (see section 2.2).

Synthesis of nor-mephedrone succinate (M4)

Nor-mephedrone conjugated with succinic acid was qualitatively synthesized by

mixing 100 µL of nor-mephedrone at 100 µg/mL with 100 mg of succinic anhydride in

acetone. The mixture was heated at 60 ºC 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-

QTOFMS analysis using the experimental conditions described above (see section 2.2).

Urine samples

Urine samples were obtained at pre-dose and after 4 hours from two volunteers

that were administered with a single oral dose of 200 mg of mephedrone in the IMIM

Clinical Research Unit. Subjects were two Caucasian healthy male recreational users of

psychostimulants and hallucinogens (ages 29 and 36 years old). Both were extensive-

intermediate CYP2D6 metabolizers.. They were free of any medicine or drugs of abuse.

A rapid urine test for main drugs of abuse was negative before administration. Subjects


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signed an informed consent and the protocol was authorized by the local ethical

committee (CEIC-PSMAR, ref. IMIMFTCL/MEF/1, 2013/5045).

Sample treatment

1 mL of urine sample was diluted two-fold with Milli-Q water and centrifuged at

10,000 r.p.m. for 7 minutes. After that, 20 µL were directly injected in the LC-QTOF

MS system.


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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 (Bijlsma et al., 2011;

Deventer et al., 2009). 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 (Bijlsma

et al., 2011; Deventer et al., 2009). 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 since all the ions with abundances higher than 20% still

preserve the nitrogen (Table 1). Two losses of radical CH3• were observed without

losing the amine. This behavior can be considered as exceptional since 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 rearrangement previous to the

fragmentation (Figure 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 since

the ion at m/z 148 is more abundant than the ion at m/z 145 (Figure 1, bottom).


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Detection of mephedrone metabolites

The strategy applied in this study (dilution of the sample, acquisition in UHPLC-QTOF

MS, and processing by MetaboLynx software) to urine samples collected before and

after administration of 200 mg of mephedrone allowed for the detection of the parent

compound and 10 metabolites (Table 1).

The mass accuracy obtained provided only 1 feasible formula for each metabolite at the

selected narrow-mass window (2 mDa). Among the 10 metabolites found, four were

detected only in the positive ionization mode, similar to the behavior observed for

mephedrone. Six of them were also detected in negative ionization mode (Table 1)

suggesting the presence of an acidic moiety.

The product ion scan of each detected metabolite was acquired and the results are

shown in Table 1. Based on the comparison between these results and the CID behavior

of mephedrone (see section 3.1), a putative structure for each metabolite was established

(see section 3.3).

In those cases in which a phase II metabolite was predicted (M4, M8, M9 and M10) or a

single abundant product ion was obtained (M2 and M7), a MS3 experiment was

performed by selecting the product ion generated in-source at high cone voltage and

acquiring the product ion scan of this in-source fragment. This allowed more knowledge

to be garnered or to obtain additional relevant information about the unconjugated

metabolite. The results of these experiments are shown in Table 1.

Mass spectrometric results for mephedrone metabolites

Metabolite M1


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The molecular formula obtained for metabolite M1 (C10H13NO) suggested the loss of a

methyl group from mephedrone. Since N-demethylation is a common metabolic

pathway of N-methyl stimulants, N-demethyl mephedrone was postulated as 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 Figure 1).

The structure of M1 was confirmed by comparison with the reference material (Figure

2, bottom).

Metabolite M2

The molecular formula of M2 (C10H11NO3) indicated a double oxidation from M1.

Additionally, 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

Figure 2) suggesting a proximity between the amine and the acidic groups. Additionally,

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


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dominated by the tropylium ion (m/z 91.0552) and its subsequent fragmentation

(Supplemental Figure 2). Additionally, 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

potential structure for M2.

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 behavior is similar to the one observed

for ephedrine derivatives which have a hydroxyl group in C1. Therefore, the reduction

of the ketone moiety would hamper the rearrangement postulated for mephedrone

decreasing the number of N-containing product ions (Supplemental Figure 3).

The structure of this metabolite as 1-dihydro mephedrone was confirmed by

comparison with synthesized material (Figure 2, middle).

Metabolite M4

Metabolite M4 exhibited a molecular formula containing three carbon atoms more than

mephedrone (C14H17NO4). This fact suggested that M4 was conjugated by phase II

metabolism. A diacetyl metabolite with the same formula was reported in the in vitro

metabolism of mephedrone (Khreit et al., 2013). However, M4 showed ionization in


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both positive and negative mode (Table 1) indicating the presence of an acidic moiety

in M4. This result is in disagreement with the diacetyl metabolite.

The product ion spectrum of M4 was one of the most complex among the detected

metabolites (Figure 3a). It showed that the molecule could be divided in two parts: one

fragment at m/z 164 (C10H14NO) and the other at m/z 101 (C4H5O3). The fragment

C10H14NO has the same molecular formula than M1 i.e. nor-mephedrone. Therefore, the

fragment at m/z 101 provided information about the phase II metabolite. The molecular

formula was in agreement with a succinyl conjugate. The product ion spectra could be

explained by this conjugation as shown in Figure 3c.

The structure of M4 as N-succinyl nor-mephedrone was confirmed by comparison

with synthetic material as shown in Figure 3b.

Metabolite M5

Metabolite M5 showed only positive ionization with a molecular formula of C10H15NO.

This formula suggested a demethylation followed by a reduction of the keto function.

Therefore, 1-dihydro nor-mephedrone was selected as most feasible candidate for M5.

This hypothesis was supported by the product ion spectrum. Similar to M3, the absence

of the ketone functionality seems to hamper the rearrangement observed in mephedrone.

In agreement with that, the product ion spectrum of M5 was dominated by subsequent

neutral losses of water and ammonia (Supplemental Figure 4).

The proposed structure was confirmed by the reduction of nor-mephedrone as can be

seen in Figure 2 (top).


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Metabolite M6

The molecular formula obtained for M6 (C11H15NO2) indicated a hydroxylation from

mephedrone. Since mephedrone contains several potential hydroxylation sites, the

product ion spectrum was used to propose the most feasible one.

The product ion spectrum showed the two expected losses of water: the one coming

from the ketone observed in most of mephedrone metabolites and the other coming

from the incorporated hydroxyl group. Additionally, an abundant neutral loss of

formaldehyde was also observed (see Table 1 and Supplemental Figure 5). This

neutral loss of 30 Da has been reported for hydroxymethyl metabolites in compounds

with stable rings like steroids (Pozo et al., 2008). Therefore, 4’-hydroxymethyl-

mephedrone was proposed for M6 in agreement with previously published studies

(Meyer et al., 2010; Khreit et al., 2013; Pedersen et al., 2013; Martinez-Clemente et al.,

2013).

Metabolite M7

The molecular formula of M7 (C11H13NO3) suggested a double oxidation from

mephedrone. Similar to M2, the oxidation of a methyl group to carboxylic acid seems to

be the most feasible mechanism to obtain M7. This fact was also supported by the

negative ionization observed for M7 (Table 1).

In contrast to M2, the product ion spectrum of M7 did not show the ion C8H7O (m/z

119.0500) indicative of an intact benzylic area. The spectrum of M7 was dominated by

losses of H2O, CO2 and CH3•. These losses could be explained after the rearrangement

described for mephedrone if the structure of M7 is 4’-carboxy-mephedrone


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(Supplemental Figure 6) as previously reported (Khreit et al., 2013; Pedersen et al.,

2013; Martinez-Clemente et al., 2013).

Metabolite M8

M8 showed a molecular formula of C17H23NO8 which implied an increase of C6H8O6

from hydroxyl-mephedrone. This increase is associated with a glucuronide moiety.

Additionally, M8 was detected in both positive and negative ionization modes (Table 1).

Therefore hydroxyl-mephedrone-3-O-glucuronide was proposed as feasible structure

for M8.

The product ion spectrum confirmed the presence of the glucuronide moiety by the

typical loss of 176 Da in positive ionization mode and the occurrence of the ion at m/z

113 in negative ionization mode associated with the glucuronide functionality (Fabregat

et al., 2013). Additionally, the product ion spectrum helped in the assignation of the

hydroxylation site (Supplemental Figure 7). The product ion spectrum of the in-source

fragment at m/z 194 (produced after the neutral loss of the glucuronide and

corresponding to the unconjugated metabolite) showed an abundant ion at m/z 119.0497

(Table 1) corresponding to CH3-Ph-CO+. As commented for other metabolites, this ion

is indicative of unaltered benzylic zone in M8. Therefore, C3 seems to be the most

suitable hydroxylation site. Similarly to M2 in which a carboxylic acid was proposed in

C3, M8 also showed the ions corresponding to the loss of CO and the subsequent gain of

a water molecule (Supplemental Figure 7) supporting the assignation. Therefore, a C3

hydroxylation previous to glucuronidation is proposed as the most feasible metabolic

pathway to obtain M8.


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Metabolite M9

The molecular formula for M9 (C17H21NO9) also suggested a glucuronide conjugate. In

that case, the phase I metabolite seemed to be a double oxidized metabolite.

The product ion spectrum of M9 after the loss of glucuronide moiety was identical to

the one observed for M7 (see Table 1 and Supplemental Figure 8). Therefore, the

glucuronide conjugate of 4’-carboxy-mephedrone was proposed as structure of M9.

Based on the structure of M7, N-glucuronidation seems to be the most feasible

metabolic pathway to obtain M9 (4’-carboxy-mephedrone-N-glucuronide).

Metabolite M10

Similarly to M8 and M9, the molecular formula of M10 (C16H20NO8) indicated a

glucuronidation. In that case, the molecular formula of the phase I metabolite produced

after the loss of the glucuronide moiety (C10H13NO2) corresponded to a hydroxylated

metabolite of nor-mephedrone.

The product ion spectrum of M10 exhibited an abundant ion at m/z 119.0478 (C8H7O).

As described for other metabolites like M2 and M8, this ion is indicative of an intact

aromatic area (Supplemental Figure 9). Following the considerations described for

these metabolites, hydroxylation in C3 of nor-mephedrone and subsequent

glucuronidation was proposed as potential metabolic pathway to obtain M10 (hydroxyl

nor-mephedrone-3-O-glucuronide). The C3-hydroxylated metabolite precursor of

M10 might be also proposed as intermediate metabolite in the generation of M2 (Figure

4).


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DISCUSSION

The study of mephedrone metabolism by combination of UHPLC-QTOF MS and

MetaboLynx after dilution of the sample allowed for the detection of mephedrone and

10 metabolites in human urine. The metabolic pathway proposed based on our results is

summarized in Figure 4.

The accurate mass measurements played a critical role to properly interpret the

fragmentation of mephedrone and to propose suitable structures for detected metabolites.

As an example, mephedrone and most of its metabolites show a product ion with

nominal m/z 119. In a previous report, this ion was assigned to CH3PhCO+ suggesting a

cleavage of the C1-C2 bond of the molecule (Martinez-Clemente et al., 2013). However,

accurate mass measurements revealed that it corresponds to a formula of C9H11

indicating that the expected cleavage is not produced. In contrast, several metabolites

produced the ion CH3PhCO+, which was confirmed by the subsequent loss of CO and

the ion at m/z 109 corresponding to [CH3PhCO – CO + H2O]+. The presence of this ion

would imply a structural change in the surroundings of the nitrogen atom. Therefore,

discerning between both C9H11 and C8H7O formulae for the ion at m/z 119 was found to

be compulsory 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 and in the benzylic carbon and oxidation of C3 and

the benzylic methyl to carboxylic acid (Figure 4). Our data confirmed the presence of

some compounds that have been previously described as mephedrone metabolites in

humans or other species (e.g. M1, M3, M5, M6, M7 and M9) (Meyer et al., 2010;

Khreit et al., 2013; Pedersen et al., 2013; Martinez-Clemente et al., 2013). However, it


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is remarkable that structures for M2, M4, M8 and M10 have been reported for the first

time in this study.

Regarding phase II metabolism, three metabolites conjugated with glucuronic acid (M8,

M9 and M10) were found. More surprising was the detection of a conjugate with

succinic acid (M4). To the best of our knowledge, this is the first reporting of such

conjugate in human urine. Succinylation in drug development falls into the prodrug

category. Succinic acid derivatives are attached to target drugs to produce more

desirable physical and biological properties, with the goal that the resulting conjugates

are degraded by cellular enzymes to release the target drugs in vivo. In the context of

physiologic compounds, biogenic amines like octopamine, dopamine, and serotonin are

typically deactivated by acetylation in insects, nematodes, and other invertebrates.

Recently, succinylation, in addition to acetylation, has been reported for octopamine and

tyramine ascarosides in nematodes opening the question as to which extend this

metabolic reaction may represent a general pathway of biogenic amines metabolism

(Artyukhin et al., 2013). In the context of the present study succinic acid was

conjugated to nor-mephedrone. The hydrolysis of the conjugate would free nor-

mephedrone, a metabolite with unknown pharmacological activity but presumably

lower than the corresponding to mephedrone.

The structure of four of the detected metabolites (M1, M3, M4 and M5) has been

confirmed by comparison with synthesized material. All four were correctly assigned

based on their mass spectrometric behavior. The synthesis of the rest of metabolites

would be required in order to ultimately confirm their structure.

In vitro studies suggest that mephedrone metabolic disposition is regulated by the

highly polymorphic isoenzyme of cytochrome P450, CYP2D6 (Pedersen et al., 2013).

The quantification of metabolites identified in this report is of importance since it is


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unknown the in vivo relevance of CYP2D6 polymorphism in mephedrone disposition

and which metabolic pathways are regulated by this isoenzyme in order to interpret

metabolic profiles in the context of forensic toxicology.

In the near future, it would be highly recommended to evaluate the behavior of

unchanged mephedrone and the 10 reported metabolites in urine samples collected for a

larger population ideally after administration of different doses, in order to determine

which should be the most appropriate biomarkers when investigating drug use.


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AUTORSHIP CONTRIBUTIONS:

Participated in research design: de la Torre, Hernandez, Farré, Sancho, Pozo

Conducted experiments: Ibañez, Lahoz- Beneytez, Pozo, 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


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FOOTNOTES

Financial support: Spanish Ministry of Economy and Competitiveness [Ref CTQ2012-

36189], Ministry of Health [ISCIII PI11/01961], DIUE de la Generalitat de Catalunya

[2014 SGR680]. and Generalitat Valenciana [research group of excellence PROMETEO

II/2014/023; ISIC 2012/016]. Supported in part by grants from Instituto de Salud Carlos

III-FEDER [FIS PI11/0196, RTA RD12/0028/0009 and Rio Hortega fellowship ISC-

III-CM13/00016].

Reprint requests: [email protected]


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FIGURE LEGENDS

Figure 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).

Figure 2. MS/MS spectra (at 20 eV) of (a) metabolites found in urine sample, and (b)

synthesized material/reference standards.

Figure 3. MS/MS spectra at 20eV in positive ionization mode (top) and negative

ionization mode (bottom) for metabolite 4 (N-succinyl nor-mephedrone) in (a) urine and

(b) synthesized reference standard. (c) proposed fragmentation pathway for this

metabolite.

Figure 4. Metabolic pathway proposed for mephedrone in human.


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TABLES

Table 1. Mass spectrometric results for the detected metabolites.

Metabolite Rt (min)

Ionization mode

m/z Error (mDa)

Formula Precursor ion

Product ion

Abundance (%)

Error (mDa)

Formula

Mephedrone 4.16 ESI+ 178.1231 -0.1 C11H16NO 178 160.1125 45 -0.1 C11H14N 147.0815 2 0.5 C10H11O 145.0889 100 -0.2 C10H11N 144.0813 22 0 C10H10NO 130.0663 2 0.6 C9H8N 119.0862 15 0.1 C9H11 117.0704 2 0 C9H9 91.0548 4 0 C7H7 79.0546 2 -0.2 C6H7 M1 3.87 ESI+ 164.1078 0.3 C10H14NO 164 147.0803 8 -0.7 C10H11O 146.0972 50 0.2 C10H12N 131.0737 100 0.2 C9H9N 130.0662 20 0.5 C9H8N 119.0862 22 0.1 C9H11 117.0707 5 0.3 C9H9 91.0550 8 0.2 C7H7 79.0547 2 -0.1 C6H7 M2 3.84 ESI+ 194.0823 0.6 C10H12NO3 194 138.0666 12 -1.5 C8H10O2 119.0497 100 0 C8H7O 109.0654 5 0.1 C7H9O 91.0547 20 -0.1 C7H7 119 109.0657 10 0.4 C7H9O 91.0546 100 -0.2 C7H7 77.0391 5 0 C6H5

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65.0387 8 -0.4 C5H5 ESI- 192.0663 0.2 C10H10NO3 192 148.0763 100 0.1 C9H10NO 146.0603 8 -0.3 C9H8NO 91.0546 32 -0.2 C7H7 M3 3.92 ESI+ 180.1387 -0.1 C11H18NO 180 162.1276 100 -0.7 C11H16N 147.1028 70 -2.0 C10H13N 146.0971 12 0.1 C10H12N 131.0861 32 0 C10H11 129.0706 8 0.2 C10H9 105.0706 7 0.2 C8H9 91.0547 18 -0.1 C7H7 70.0655 5 -0.2 C4H8N M4 7.38 ESI+ 264.1234 -0.2 C14H18NO4 264 246.1115 8 -0.2 C14H16NO3 228.1024 8 -0.1 C14H14NO2 186.0923 9 0.4 C12H12NO 164.1084 18 0.5 C10H14NO 147.0815 40 0.5 C10H11O 146.0971 100 0.1 C10H12N 131.0725 10 -1.0 C9H9O 126.0555 62 0 C6H8NO2 119.0860 58 -0.1 C9H11 101.0238 22 -0.1 C4H5O3 98.0617 7 1.1 C5H8NO 164 146.0971 48 0.1 C10H12N 131.0725 100 -1.0 C9H9O 119.0859 28 -0.2 C9H11 117.0705 5 0.1 C9H9 91.0548 8 0 C7H7 67.0554 2 0.6 C5H7 ESI- 262.1085 0.6 C14H16NO4 262 244.0972 78 -0.2 C14H14NO3




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226.0847 42 -2.1 C14H12NO2 200.1071 100 -0.4 C13H14NO 162.0932 18 1.3 C10H12NO 133.0639 18 -1.4 C9H9O 99.0079 38 -0.3 C4H3O3 98.0271 25 2.9 C4H4NO2 M5 3.84 ESI+ 166.1231 -0.1 C10H16NO 166 148.1122 72 -0.4 C10H14N 131.0861 100 0 C10H11 116.0627 28 0.1 C9H8 115.0545 12 -0.3 C9H7 105.0703 5 -0.1 C8H9 91.0548 60 0 C7H7 56.0488 2 -1.2 C3H6N M6 2.61 ESI+ 194.1178 0.3 C11H16NO2 194 176.1057 2 -1.8 C11H14NO 158.0961 100 -0.9 C11H12N 146.0962 100 -0.6 C10H12N 133.0651 8 -0.2 C9H9O 131.0728 43 -0.7 C9H9N 105.0696 12 -0.8 C8H9 M7 1.19 ESI+ 208.0973 -0.1 C11H14NO3 208 190.0879 5 1.1 C11H12NO2 172.0762 35 0 C11H10NO 146.0968 81 -0.2 C10H12N 144.0813 18 0 C10H10N 131.0735 100 0 C9H9N 130.0656 8 -0.1 C9H8N 105.0702 20 -0.2 C8H9 ESI- 206.0812 -0.5 C11H12NO3 206 162.0914 100 -0.5 C10H12NO 162 160.0766 62 0.4 C10H10NO 147.0696 20 1.2 C9H9NO 84.0438 8 -0.1 C4H6NO




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M8 6.62 ESI+ 370.1509 0.7 C17H24NO8 370 209.1169 12 -0.9 C12H17O3 194.1176 100 -0.5 C11H16NO2 191.1075 22 0.3 C12H15O2 178.1234 8 0.2 C11H16NO 160.1121 32 -0.5 C11H14N 119.0497 68 0 C8H7O 194 161.0818 10 0.7 C10H11NO 148.1096 8 -0.3 C7H16O3 119.0502 100 0.5 C8H7O 109.0656 8 0.3 C7H9O 91.0554 35 0.6 C7H7 79.0545 5 -0.3 C6H7 ESI- 368.1358 1.1 C17H22NO8 368 193.0335 62 -1.3 C6H9O7 157.0137 18 0 C6H5O5 131.0350 38 0.7 C5H7O4 113.0239 100 0.4 C5H5O3 103.0022 40 -0.9 C3H3O4 72.9917 32 -0.9 C2HO3 M9 1.27 ESI+ 384.1302 0.7 C17H22NO9 384 208.0982 100 0.8 C11H14NO3 190.0862 22 -0.6 C11H12NO2 172.0763 58 0.1 C11H10NO 146.0968 32 -0.2 C10H12N 208 190.0853 5 -1.5 C11H12NO2 172.0763 25 0.1 C11H10NO 146.0972 82 0.2 C10H12N 131.0737 100 0.2 C9H9N 105.0707 22 0.3 C8H9 ESI- 382.1128 -1.0 C17H20NO9 382 206.0812 100 -0.5 C11H12NO3 162.0915 58 -0.4 C10H12NO 113.0234 72 -0.5 C5H5O3




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85.0280 22 -1.0 C4H5O2 206 162.0923 100 0.4 C10H12NO 160.0762 8 -0.9 C10H10NO 77.0386 5 -0.5 C6H5 M10 5.71 ESI+ 356.1346 0.1 C16H22NO8 356 180.1028 100 0.3 C10H14NO2 162.0919 72 0 C10H12NO 119.0478 45 -1.9 C8H7O 180 149.0974 22 0.8 C10H13O 131.0864 100 0.3 C10H11 107.0867 18 0.6 C8H11 91.0543 50 -0.5 C7H7 ESI- 354.1185 -0.4 C16H20NO8 354 193.0364 38 1.1 C9H7NO4 113.0237 42 -0.2 C5H5O3 85.0303 18 1.3 C4H5O2




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MASS SPECTROMETRIC EVALUATION OF …dmd.aspetjournals.org/content/dmd/early/2014/12/02/dmd.114.061416...dmd#61416 1 mass spectrometric evaluation of mephedrone in vivo human metabolism: