Mechanism of aromatic hydroxylation of lidocaine at a Pt electrode under acidic conditions

Article abstract of DOI:10.1016/j.electacta.2016.12.089

Aromatic hydroxylation reactions, which are mainly catalyzed by cytochrome P450 (CYP) enzymes in vivo, are some of the most important reactions of Phase I metabolism, because insertion of a hydroxyl group into a lipophilic drug compound increases its hydrophilicity and prepares it for subsequent Phase II metabolic conjugation reactions as a prerequisite to excretion. Aromatic hydroxylation metabolites of pharmaceuticals may be obtained through various synthetic and enzymatic methods Electrochemical oxidation is an alternative with advantages in terms of mild reaction conditions and less hazardous chemicals. In the present study, we report that aromatic hydroxylation metabolites of lidocaine can be readily obtained electrochemically under aqueous acidic conditions at platinum electrodes. Our results show that the dominant N-dealkylation reaction can be suppressed by decreasing the solution pH below 0.5 resulting in selective 3-hydroxylidocaine, which is an in vivo metabolite of lidocaine. Experiments in ¹⁸O labelled water indicated that water is the primary source of oxygen, while dissolved molecular oxygen contributes to a minor extent to the hydroxylation reaction.

Full text of DOI:10.1016/j.electacta.2016.12.089

Accepted Manuscript
Title: Mechanism of aromatic hydroxylation of lidocaine at a
Pt electrode under acidic conditions
Author: Turan Gu¨l Rainer Bischoff Hjalmar P. Permentier
PII:
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Mechanism of aromatic hydroxylation of lidocaine at a Pt electrode under acidic
conditions
Turan Gül1, Rainer Bischoff1, Hjalmar P. Permentier1,2*
1Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen,
Groningen, The Netherlands
2Interfaculty Mass Spectrometry Center, University of Groningen, Groningen, The Netherlands
*Corresponding author: Hjalmar Permentier, Interfaculty Mass Spectrometry Center, University of
Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. Tel. +31-50-363-
3262, Fax +31-50-363-7582, e-mail: h.p.permentier@rug.nl

Abstract
Aromatic hydroxylation reactions, which are mainly catalyzed by cytochrome P450 (CYP)
enzymes in vivo, are some of the most important reactions of Phase I metabolism, because insertion
of a hydroxyl group into a lipophilic drug compound increases its hydrophilicity and prepares it for
subsequent Phase II metabolic conjugation reactions as a prerequisite to excretion. Aromatic
hydroxylation metabolites of pharmaceuticals may be obtained through various synthetic and
enzymatic methods. Electrochemical oxidation is an alternative with advantages in terms of mild
reaction conditions and less hazardous chemicals. In the present study, we report that aromatic
hydroxylation metabolites of lidocaine can be readily obtained electrochemically under aqueous
acidic conditions at platinum electrodes. Our results show that the dominant N-dealkylation
reaction can be suppressed by decreasing the solution pH below 0.5 resulting in selective 3-
hydroxylidocaine, which is an in vivo metabolite of lidocaine. Experiments in 18O labelled water
indicated that water is the primary source of oxygen, while dissolved molecular oxygen contributes
to a minor extent to the hydroxylation reaction.
Key words
-
-
-
-
-
Drug metabolism
Aromatic hydroxylation
Pt-oxide layer
Electrochemical synthesis
18O labeled water

1. Introduction
Aromatic hydroxylation reactions of pharmaceuticals have been extensively studied due to
their importance in the biotransformation of drugs and in wastewater treatment for the removal of
drug residues [1–3]. In vivo, the hydroxylation reaction is mainly catalyzed by cytochrome P450
(CYP) enzymes in the liver, where it is an important reaction of Phase I metabolism, resulting in
the bioactivation or detoxification of drug molecules [4–6]. Insertion of a hydroxyl group into a
lipophilic drug compound increases solubility so that drugs can be more easily excreted, either
directly or after conjugation with more polar moieties, such as sulfates or glucuronides [7].
In order to study hydroxylated drug metabolites and assess their toxicity during the drug
development process, hydroxylation of drug substrates has been performed by a wide variety of
enzymatic, organic and electrochemical synthesis methods. In the enzymatic conversions purified
or enriched CYP enzymes are used to insert an oxygen atom into the C-H bond of an aromatic
substrate [8–10]. In organic synthesis methods oxidizing reagents such as peroxides or peroxyacids
and metal catalysts are employed to generate hydroxyl radicals (Fenton and Haber-Weiss reactions)
which readily react with aromatic substrates to form hydroxylation products [2,11–14]. Compared
to enzymatic reactions, these chemical methods often require harsh reaction conditions, have low
regioselectivity and produce undesired byproducts [8,15].
Electrochemical (EC) oxidation methods have been used as alternatives to obtain aromatic
hydroxylation metabolites of drug compounds by employing electrogenerated reactive oxygen
species [16–18]. For example, the EC-assisted Fenton reaction, which produces hydroxyl radicals
from hydrogen peroxide in solution, has been used for the hydroxylation of various drug substances
including metoprolol, buspirone, promazine and 7-ethoxycoumarin [19,20]. We have extensively
studied alternative electrochemical methods for the synthesis of specific oxidation products of the
local anesthetic drug lidocaine. Lidocaine is oxidized in vivo to the aromatic hydroxylation
products 3- and 4-hydroxylidocaine, and can also undergo benzylic hydroxylation, N-dealkylation
and N-oxidation reactions [21,22]. By adjusting the electrochemical parameters, a certain degree
of product selectivity can be achieved. Direct electrochemical oxidation of lidocaine in aqueous
solution produces N-dealkylation and N-oxidation but no hydroxylation products. Aromatic
hydroxylation was only observed at potentials higher than 3 V in the presence of 1% water resulting
mainly in 4-hydroxylidocaine [23]. Oxidation in the presence of 1% hydrogen peroxide at 3 V led

to both 3- and 4-hydroxylidocaine, but no N-oxide was formed [24]. In the latter reaction, we
proposed that hydrogen peroxide was electrocatalytically activated on the platinum electrode
forming platinum-oxo species capable of direct insertion of an oxygen atom into the C-H bond of
the aromatic ring [24].
The role of platinum-oxide layers in electrochemical aromatic hydroxylation reactions has
been extensively studied in the literature. It has been reported that oxide layers are formed on the
Pt electrode surface under strong acidic conditions [25–29]. Conway et al. showed that a water
molecule binds to the electrode surface and that a platinum oxide species is formed following the
irreversible removal of one electron and one proton [27,28,30]. This Pt-oxide layer is claimed to
increase electron transfer efficiency [31] thereby facilitating electrochemical oxidation of many
organic substances. For example, oxidation of aromatic compounds, including benzene and phenol,
has been achieved in aqueous acidic solutions (e.g. 0.5 M H2SO4) when oxide layers are present
on the Pt electrode surface [32–34].
We showed recently that N-dealkylation of lidocaine is favored under basic conditions, while
this reaction was completely suppressed at pH values below 0.5 in the presence of trifluoroacetic
acid (TFA) [35]. In addition, we observed that aromatic hydroxylation products were readily
produced under these conditions. In the present study we attempt to elucidate the mechanism of
the aromatic hydroxylation reaction in the presence of TFA at platinum electrodes, the role of
platinum-oxide layers and notably to define the source of oxygen.
2. Materials and Methods
2.1. Reagents
18
Lidocaine (L7757), trifluoroacetic acid (TFA, T6508) and H2 O (329878) were purchased
from Sigma-Aldrich (Zwijndrecht, The Netherlands). Formic acid (HCOOH, 94318) and
acetaminophen (00370) were purchased from Fluka and ultra-pure HPLC grade acetonitrile (ACN,
01203502) was purchased from Biosolve (Valkenswaard, The Netherlands). Sulfuric acid (H2SO4,
1007311) was purchased from Merck Millipore (Amsterdam, The Netherlands). Ultrapure water
was obtained from a Milli-Q Advantage A10 Water Purification system (Millipore Corp., Billerica,
MA, USA).

2.2. Electrochemical measurements
Constant potential measurements were performed with an Antec ROXY potentiostat (Antec
Leyden, Zoeterwoude, The Netherlands) controlled by Antec Dialogue software. Cyclic
voltammetry (CV) measurements were performed with an Autolab PGSTAT204 potentiostat
(Metrohm, Schiedam, The Netherlands) controlled by Metrohm Nova software. Unless otherwise
stated, electrochemical reactions were performed in a one-compartment three-electrode cell in
which the working electrode was a Pt coil (P3640/88, Fischer Scientific, Landsmeer, The
Netherlands) and the auxiliary electrode a platinum wire (MW-4130, BASi, West Lafayette, IN,
USA). During CV measurements, a Ag/AgCl (in 3 M NaCl) reference electrode was used, while
for constant potential experiments a silver wire pseudo-reference electrode (MF-2017, BASi) was
used to avoid chloride contamination during electrochemical reactions. The potential shift of the
Ag wire pseudo reference compared with the Ag/AgCl 3 M KCl reference electrode is -0.4 V,
measured in an aqueous 0.1 M NaCl solution with 10 mM potassium hexacyanoferrate. All
electrochemical experiments were performed at ambient temperature. For deaeration, nitrogen, or
helium was bubbled via a sparge tube (MW-4145, BASi). Working electrodes were cleaned by
sonicating in ethanol followed by drying with nitrogen.
For strong acidic conditions (pH 0.5, measured with a S20 SevenEasy pH meter (Mettler-
Toledo, Schwerzenbach, Switzerland) using a pH electrode), 10 M lidocaine solutions were
prepared either in ACN/H2O/TFA (90/5/5) or in ACN/TFA (95/5). For CV measurements, a 1 M
TFA solution in water was prepared. Unless otherwise stated, 3 mL solutions of lidocaine were
oxidized for 30 min in a one-compartment three electrode cell. For oxygen-free conditions, a three-
neck round-bottom flask was used and the working (Pt coil), auxiliary (Pt coil) and reference (Ag
wire) electrodes were fixed each into a separate neck of the flask by using a rubber septum. For
purging with nitrogen and sampling, two different sparge tubes were also inserted through the
septa. The necks of the flask were sealed and air was removed by purging with nitrogen gas for 30
min while simultaneously applying vacuum (Supplementary Figure S1). In order to remove
dissolved oxygen from the lidocaine solutions, the solvent bottle was sealed and purged with
helium gas for 15 min before introduction into the three-neck flask. After electrochemical
oxidation, samples were diluted with water containing 100 M acetaminophen as internal standard

(IS). The final samples for LC-MS analysis contained 1  lidocaine (based on the initial
concentration) and 10 M acetaminophen.
2.3. LC-MS analysis
LC-MS/MS analyses in the selected reaction monitoring (SRM) mode were carried out on an
HPLC system with an Accela Autosampler and a Surveyor Pump coupled to a TSQ Quantum AM
triple quadrupole mass spectrometer (Thermo Finnigan, San José, CA, USA) with an electrospray
ionization interface in the positive mode. The MS parameters for the TSQ Quantum AM were as
follows: spray voltage 3500 V, auxiliary gas pressure 20 (arbitrary unit), sheath gas pressure 40
(arbitrary unit), capillary temperature 350 ºC, tube lens offset 90 V, skimmer offset -0 V, scan
range m/z 100-300 (scan time 1 s, Q1 peak width 0.70 amu FWHM). The SRM transitions selected
for lidocaine and its metabolites were: lidocaine: 235/86, N-dealkylation product: 207/58, aromatic
hydroxylation products: 251/86. The transition 152/110 was selected for acetaminophen. SRM
measurements were performed with a dwell time of 100 ms for each transition and Q3 peak widths
of 0.70 amu FWHM.
The LC separation of lidocaine and its products was performed with a C18 reversed-phase
column (GraceSmart RP 18, 5 m particle size, 2.1×150 mm; Grace Davison, Lokeren, Belgium)
at a flow rate of 250 L/min. Solvent A was H2O with 0.1% formic acid, while solvent B was ACN
with 0.1% formic acid. A linear gradient was applied starting from 5% to 95% solvent B in solvent
A over 11 min, which was held for 1 min. Solvent B was decreased rapidly to 5% in 20 s and the
column was re-equilibrated at 5% solvent B for 4 min. Acetaminophen was used to normalize the
peak areas of lidocaine and its oxidation products, resulting in normalized product yields.
3. Results and Discussion
3.1. Aromatic hydroxylation reactions of lidocaine under strong acidic conditions
As previously reported, the electrochemical N-dealkylation reaction of lidocaine is
completely suppressed at pH values below 0.5 [35]. Unexpectedly, under these strongly acidic
conditions aromatic hydroxylation metabolites of lidocaine are produced instead. Since aromatic
hydroxylation of lidocaine has not been reported before at potentials as low as 1.5 V, we
investigated this reaction in more detail. For this purpose, a solution of 10 M lidocaine was
prepared in ACN/TFA/H2O (90:5:5) and oxidized at 1.5 V for 30 min. Samples were analyzed by
LC-MS/MS showing the generation of 3- and 4-hydroxylidocaine (Figure 1). The peak at 2.2 min

was assigned to 3-hydroxylidocaine using a synthetic standard, while the other peak at 1.9 min was
assigned to 4-hydroxylidocaine based on literature data [36]. The ratio of the LC-MS peak areas of
3- and 4-hydroxylidocaine is 10:1. No other reaction products of lidocaine, including benzylic
hydroxylation, N-oxide formation, and dihydroxylation were observed.
3.2. Investigation of the oxygen source
In order to gain a better understanding of the reaction mechanism, we first investigated
whether water could serve as the source of oxygen and performed the reaction in 18O-labelled water
18
(97% O atom). A solution of 10 M lidocaine was prepared in ACN/TFA/18OH2 (90:5:5) and
oxidized at 1.5 V for 90 min in a three-neck round-bottom flask while purging with nitrogen gas.
The LC-MS/MS results of collected samples showed that approximately 86% of the generated 3-
hydroxylidocaine contained 18O (Figure 2a). The remaining 14 % of 3-hydroxylidocaine contained
16O which may have originated from residual unlabeled water in the reaction mixture. In support
of this finding, we found that that same experiment using a 1:1 ratio of 18OH2/16OH2 yielded 58%
of 3-hydroxylidocaine containing 16O and 42% containing 18O (Figure 2b).
We investigated other possible sources of oxygen by performing the aromatic hydroxylation
reaction in the absence of water (ACN/TFA (95:5)). The generation of 3-hydroxylidocaine under
these conditions, albeit in lower amounts (Figure 3), confirmed that there must be another source,
possibly dissolved molecular oxygen. To study this further, electrochemical hydroxylation was
performed in the absence of water under oxygen-free conditions. Lidocaine solution in ACN/TFA
(95:5) was deaerated and subsequently introduced into the reaction flask and the reaction was
carried out under continuous purging with nitrogen gas. As a control, 10 M lidocaine solution,
prepared in ACN/TFA (95:5), was purged with zero air and both solutions were oxidized at 1.5 V
for 90 min. The generation of 3-hydroxylidocaine and the consumption of lidocaine were followed
by LC-MS/MS and compared to a reaction in the presence of water by plotting product yield against
oxidation time. While very low amounts of 3-hydroxylidocaine were produced under oxygen-free
conditions (Figure 3), it was readily observed when purging with zero air. The consumption of
lidocaine under oxygen-free conditions was negligible which shows that no reaction takes place
under these conditions. Approximately 0.8 M of 3-hydroxylidocaine was detected when purging
with zero air and 1.6 M of 3-hydroxylidocaine was detected in the presence of water under
nitrogen purging, corresponding to yields of 8% and 16%, respectively. Moreover, under both

conditions the ratio of 3- to 4-hydroxylidocaine was approximately 10 to 1 as was also observed in
the experiments shown in Figure 2. Taken together these results indicate that dissolved molecular
oxygen may also serve as oxygen source. We assume that molecular oxygen participates in the
aromatic hydroxylation reaction after its reduction to water at the counter electrode (Eq. 1), a
reaction that is catalyzed by metal oxide electrodes [37], although we cannot exclude that other
reactive oxygen intermediates derived from oxygen play a role. Since the counter electrode and
working electrode compartments are not separated, water may diffuse to the working electrode and
thus ultimately serve as the source of oxygen.
3.3. Investigation of the aromatic hydroxylation mechanism
The aromatic hydroxylation reaction of lidocaine in the presence of water is thought to
proceed through the formation of hydroxyl radicals upon electrochemical oxidation of water
[17,38–40]. Comninellis et al. reported that oxidation of water at metal electrodes generates
physically adsorbed hydroxyl radicals (Eq. (3)) [39–41]. Organic compounds are presumed to be
oxidized by either the physically adsorbed hydroxyl radicals (Eq. (4) and (5)) or by chemically
adsorbed oxygen (Eq. (6)) or by a combination of these pathways [40].
O2 + 4H+ + 4 e‒  2H2O
(1)
(2)
Pt + xH2O  PtOx + 2xH+ + e‒
.
PtOx + H2O  PtOx( OH) + H+ + e‒
(3)
(4)
.
.
.
PtOx( OH) + RH  PtOx( OH)y-1 + H2O + R
.
.
.
PtOx( OH)y-1 + R  PtOx( OH)y-2 + ROH
PtOx+1 + RH  PtOx + ROH
(5)
(6)
Since previous work has shown that oxide layers on the Pt surface are involved in
hydroxylation reactions, we performed experiments in 18OH2 to produce a Pt-18O layer, and
checked whether 18O from this layer was subsequently incorporated into lidocaine. Initially, cyclic
voltammetry between -0.1 V and 1.5 V at a 100 mV/s scan rate was performed in the presence of
a 1 M aqueous TFA solution using a Pt coil working electrode to follow consecutive oxide layer
formation and removal. The cyclic voltammograms indicated that oxide layers were formed at

potentials above 1.0 V and removed again from the Pt electrode surface between 0.3 V and 0.6 V
(Figure 4) [42]. In order to grow 18O layers on the Pt surface 1 M H2S16O4 was prepared in 18OH2
and CV scans were performed by following the protocol reported in the literature [43]. The
coverage of the oxygen atoms was estimated to be equivalent to 0.5-1 monolayers (ML), based on
the charge density of ~300 C/cm2 [44]. The number of the Pt atoms on the 8 mm2 working
electrode was calculated to be 6.3 × 1013 considering a smooth surface. This corresponds to
18
approximately 0.1 nmol Pt [45] translating into 0.05-0.1 nmol oxygen atoms. The O covered
electrode was used as a working electrode to oxidize a 10 M lidocaine solution prepared in
ACN/TFA (95:5). The LC-MS/MS results of this experiment showed that the aromatic
16
hydroxylation products only contained O (16% yield, corresponding to 1.6 nmol), whereas we
would expect up to 6% 18O contribution. Assuming that the Pt-18O layer was successfully
generated, this leads to the conclusion that the oxygen atoms of the Pt-oxide layers are at most a
minor source of oxygen in the aromatic hydroxylation reaction.
Conclusions
We report that aromatic hydroxylation of lidocaine can be readily achieved electrochemically
under strongly acidic conditions (ACN/TFA) at Pt electrodes. Both water and dissolved molecular
oxygen may function as sources of oxygen. An important prerequisite is that the dominant N-
dealkylation reaction is suppressed at pH values below 0.5. Experiments in the presence of 18O-
labelled water indicate that water is the primary source of oxygen and we assume that dissolved
molecular oxygen is reduced to water at the counter electrode thus contributing to the reaction,
notably in the absence of added water. The aromatic hydroxylation mechanism in the presence of
water is assumed to proceed through activation of water to generate reactive oxygen species
particularly hydroxyl radicals.
Acknowledgements
The authors thank Dutch Technology Foundation (STW, project 11957) for providing financial
support.

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Figure Legends
Figure 1. Electrochemical conversion of lidocaine upon oxidation under strong basic or acidic
conditions. LC-MS/MS chromatograms in the SRM mode were recorded as follows: N-
dealkylation m/z 207/86, N-oxidation and aromatic hydroxylation m/z 251/86. Oxidation was
performed for 30 min at 1.5 V. Solvent conditions: a) pH 12 (ACN/NH4OH), and b) pH 0.5
(ACN/H2O/TFA). N-dealkylation and N-oxidation products were observed at pH 12 while only
aromatic hydroxylation products of lidocaine were observed at pH 0.5. Lidocaine elutes at 3.7 min
and the peak in trace a (251/86 SRM chromatogram) is due to the partial in-source oxidation of
lidocaine during the electrospray ionization process. Different intensity scales were used for
chromatograms.
Figure 2. Aromatic hydroxylation product formation upon electrochemical oxidation of lidocaine
a) in the presence of ACN/TFA/18OH2 (90:5:5), and b) ACN/TFA/18OH2/16OH2 (90:5:2.5:2.5).
Experiments were repeated two times, and the error bars indicate the standard error.
Figure 3. Effect of the presence of molecular oxygen and water on the formation of 3-
hydroxylidocaine. Experiments were repeated 5 times for the oxygen-free experiment and the error
bars show the standard error.
Figure 4. The reversible formation of Pt-oxide layers. Cyclic voltammetry experiments were
performed using a Pt electrode in 1 M TFA in water. Potentials were cycled from -0.1 V to 1.5 V
at 100 mV/s scan rate vs an Ag/AgCl (in 3 M NaCl) reference electrode.

3.7
5E4
4E4
3E4
in source oxidation
of lidocaine
m/z 251/86
pH 12
Pt electrode
30 min oxidation
a)
N-dealkylation
m/z 207/58
2.5
2E4
1E4
N-oxidation
m/z 251/86
5.4
2.0
9E5
7E5
3-hydroxy
lidocaine
m/z 251/86
pH 0.5
b)
5E5
Pt electrode
4-hydroxy
lidocaine
m/z 251/86
30 min oxidation
3E5
1E5
2.0
0
2
1
3
4
5
6
7
8
9
10
11
12
13
Time (min)
Figure 1.

10 M Lidocaine in ACN/TFA/H218O (90:5:5)
20
15
10
5
3-16OH-lidocaine
4-16OH-lidocaine
3-18OH-lidocaine
4-18OH-lidocaine
86% 18
O
a)
14% 16
O
0
0
20
40
60
80
100
Time (min)
10
8
10 M Lidocaine in ACN/TFA/H216O/H218O (90:5:2.5:2.5)
3-16OH-lidocaine
4-16OH-lidocaine
3-18OH-lidocaine
4-18OH-lidocaine
58% 16
O
6
b)
4
42% 18
O
2
0
0
20
40
60
80
100
Time (min)
Figure 2.

3-OH-lidocaine
20
15
10
5
0.8
0.6
0.4
0.2
0.0
-0.2
20
40
60
80
100
Time (min)
water free & oxygen free
water free & air purged
5% water & oxygen free
0
20
40
60
80
100
Time (min)
-5
Figure 3.

2000
1500
1000
500
water
electrolysis
Oxide
formation
Hydrogen
adsorption/desorption
-0.5
0.5
1.0
1.5
2.0
-500
Oxide
removal
Potential vs Ag/AgCl (V)
Figure 4.