Analytica Chimica Acta: Yaotian Wang, Haiyan Zhang, Mingli Chen
Analytica Chimica Acta: Yaotian Wang, Haiyan Zhang, Mingli Chen
Analytica Chimica Acta: Yaotian Wang, Haiyan Zhang, Mingli Chen
h i g h l i g h t g r a p h i c a l a b s t r a c t
a r t i c l e i n f o a b s t r a c t
Article history: Levodopa is often used to treat Parkinson’s disease. It coexists with dopamine in human fluids and is
Received 22 February 2021 electrochemically oxidized at the same potential as dopamine. Differentiating levodopa from dopamine
Received in revised form is difficult via electrochemical techniques. Taking advantage of the differences in the rate constants of
7 March 2021
levodopa and dopamine for the intramolecular cyclization reaction, we observed that the cyclization
Accepted 10 March 2021
Available online 12 March 2021
reaction of dopamine-quinone was completely suppressed under pH 4.8, while that of levodopa-quinone
occurred. The product of cyclization caused a new cathodic peak at negative potential. Its peak current
was dependent on the concentration of levodopa but not that of dopamine. As a result, we developed a
Keywords:
Catecholamines
method of detecting levodopa in the presence of dopamine with a bare glassy carbon electrode.
Dopamine © 2021 Elsevier B.V. All rights reserved.
Levodopa
Cyclization reaction
Differentiation
Electrochemistry
1. Introduction elderly all over the world. The key pathological characteristic of PD
is dopaminergic neuron degeneration in the substantia nigra [1].
Parkinson’s disease (PD) is characterized by tremor, bradyki- Levodopa can be used in the treatment of PD because it crosses the
nesia, rigidity, and postural instability [1]. It is common among the bloodebrain barrier and is subsequently converted to dopamine
(DA) by dopa decarboxylase. However, long-term levodopa use is
* Corresponding author. associated with early debilitating motor fluctuations, whereas
** Corresponding author. excessive levodopa for patients with PD causes the activation of
E-mail addresses: zhanghaiyan@ucas.ac.cn (H. Zhang), chenml@mail.neu.edu.cn
(M. Chen).
wrong neurons [2]. Thus, the detection of levodopa and DA in both
https://doi.org/10.1016/j.aca.2021.338379
0003-2670/© 2021 Elsevier B.V. All rights reserved.
Y. Wang, H. Zhang and M. Chen Analytica Chimica Acta 1157 (2021) 338379
pharmaceutical formulations and biological fluids is crucial. electrode was a platinum wire. All potentials reported in this paper
Various methods have been devised to measure the amount of were referenced to a saturated calomel electrode (SCE). Unless
levodopa and DA, including high-performance liquid chromatog- otherwise indicated, prior to each voltammetric experiment, the
raphy [3e6], capillary zone electrophoresis [7e9], chem- GCE was first polished with sand paper, followed by alumina slurry
iluminescence [9], spectrophotometry [10e13], and of 1.0, 0.3 mm respectively on polishing cloth with water as the
electrochemical detection [14e23]. Electrochemical techniques lubricant. Then it was washed with aqueous and ultrasonically
have received the attention of some chemists for their low fabri- cleaned in water for 30 s.
cation cost, high sensitivity, and selectivity. In recent decades, the All experiments were carried out at room temperature. Buffer
electrochemical detection of catecholamines has been favored by solutions were prepared with acetic acid (CH3COOH) and sodium
many neurochemists. Fast-scan cyclic voltammetry has been used acetate (CH3COONa) (pH 4.4e5.6), potassium monohydrogen
to detect DA and DA-o-quinone at overoxidized polypyrrole-coated phosphate (K2HPO4) and potassium dihydrogen phosphate
carbon-fiber microelectrodes when implanted in rat brain [24]. (KH2PO4) (pH 6.0e6.8) to achieve the required pH range. The buffer
However, the detection process often encounters interference from and sample solution were deaerated with purified nitrogen for at
other common electroactive small molecules such as uric acid and least 5 min for removing oxygen prior to the beginning of a series of
ascorbic acid [15,25]. Several approaches have been applied to experiments.
suppress ascorbic acid and uric acid. One is to modify the electrode
surface with self-assembled layers that are negatively charged. The
membrane repels ascorbic acid from the surface and concentrates 3. Results and discussion
the positively charged DA. The most popular surface modification
technology is Nafion, a perfluorinated cation-exchange membrane 3.1. Voltammetric behaviors of DA and levodopa
[24,26,27].
Catecholamines cannot be easily differentiated through simple Fig. 1 shows two repeated cycles of CVs of DA and levodopa at a
electrochemical methods because of their similarity in chemical bare GCE in PBS pH 7.0 solution. At the first scan, although DA and
structure and oxidation potential. When the amine of catechol- levodopa were oxidized at the same potential, peak 1 of DA
amines is deprotonated, an o-quinone produced by electrochemical appeared at 0.18 V and that of levodopa appeared at 0.2 V. The peak
oxidation catecholamine can undergo a 1,4 (Michael) addition, current of peak 1 of DA was 27. 5 mA, and that of levodopa was
which results in a cyclization reaction and forms leucoamino- 30.9 mA. The electrochemical processes of catecholamines in
chrome [14,28,29]. Taking advantage of the differences in the aqueous solution have been pursued for more than 50 years
intramolecular cyclization rates, Hu and Fritsch applied electro- [14,28,29], as shown in Scheme 1. Peak 1 is due to the oxidation of
chemical redox cycling to differentiate DA and norepinephrine by catecholamines to their corresponding o-benzoquinone derivatives
using a model microband electrode array [14]. (1). Peak 2 corresponded to the reduction of produced o-benzo-
Electrochemically distinguishing levodopa from DA, which quinone derivatives, whereas peak 2 of DA was small and that of
coexist in regions of the central nervous system, is worth investi- levodopa disappeared. o-Benzoquinone underwent an intra-
gating. To our knowledge, an electrochemical method that differ- molecular cyclization reaction (3) to form leucoaminochrome
entiates between DA and levodopa has not yet been reported. In whose oxidation potential is lower than that of catecholamines
this work, we studied the effect of pH on the intramolecular [29]. Therefore, leucoaminochrome was rapidly oxidized by o-
cyclization rates of levodopa and DA. Under pH 4.8, the cyclization benzoquinone, producing aminochrome (4), while o-benzoquinone
of DA was suppressed while that of levodopa was obvious. Using reduced back to the original catecholamine. Catecholamine was
differential pulse voltammetry (DPV) and bare glassy carbon elec- electrochemically oxidized at the electrode again, which caused
trode (GCE), we could detect levodopa in the presence of a peak 1 to increase. The cyclization reaction led the concentration of
considerable amount of DA. o-benzoquinone near the electrode surface to decrease, which
resulted in the decrease in peak 2 and complete disappearance
2. Experimental section (Fig. 1). Moreover, at a more negative potential, another cathodic
2.1. Chemicals
2
Y. Wang, H. Zhang and M. Chen Analytica Chimica Acta 1157 (2021) 338379
Scheme 1. Scheme of electrochemical oxidation of DA and levodopa and following chemical reactions (the ECE mechanism) for DA, R ¼ H; levodopa, R ¼ COO.
peak 3 occurred at 0.296 V for DA and 0.284 V for levodopa potential difference between DA and levodopa and their products
whose currents were much higher than those of peak 2. As shown of the following chemical reaction of o-benzoquinone in Fig. 1 was
in Fig. 1, no counter peak of peak 3 was found in the first anodic so small that using electrochemical techniques could hardly
scan. However, in the second cycle, the anodic counter peak 4 distinguish them (see Fig. 2) (see Fig. 3).
appeared at 0.237 V for DA and 0.218 V for levodopa. These
results demonstrated an ECE (electron transfer, chemical reaction,
3.2. Effect of pH on the DPVs of DA and levodopa
electron transfer) mechanism in which peaks 1 and peak 2 corre-
sponded to the oxidation of catecholamine to o-benzoquinone and
The oxidation of catecholamines is the proton coupled electron
vice versa, and peaks 3 and 4 corresponded to the conversion be-
transfer reaction; therefore, their redox potentials are dependent
tween leucoaminochrome and aminochrome (4). The oxidation
on pH. We investigated the effect of pH on the DPVs of DA and
3
Y. Wang, H. Zhang and M. Chen Analytica Chimica Acta 1157 (2021) 338379
Besides pH, the DA concentration and the quiet time of DPV may
affect the stability of aminochrome from levodopa on the time
scales of electrochemical experiments. First, the effect of an in-
crease in the DA concentration on the DPVs of levodopa was
studied. Fig. 5 shows the DPVs of levodopa in pH 4.8 buffer solution
containing different concentrations of DA. Peak 2 increased as the
DA concentration increased, whereas peak 3 was almost indepen-
dent of the DA concentration, which revealed that DA did not affect
the stability of aminochrome from levodopa. Thus, interference
from DA could be eliminated in buffer solution under pH 4.8 during
the analysis of levodopa.
Fig. 4. Variations in the peak current of peak 3 versus pH for 1 mM DA and 1 mM
Fig. 6 presents that the magnitude of peak 3 rapidly increased levodopa.
with the quiet time at the expense of peak 2. The initialization
potential (Ei) of DPV was 0.6 V, which was much higher than the
oxidation potential of levodopa. Levodopa near the electrode sur-
face was rapidly oxidized at Ei. Quiet time is equal to the period of
4
Y. Wang, H. Zhang and M. Chen Analytica Chimica Acta 1157 (2021) 338379
Table 2
Determination of levodopa in capsules (n ¼ 5).
Fig. 6. DPVs of 1 mM levodopa in pH buffer 4.8 and 0.1 mol L1 KCl at different quiet 3.5. Determination of levodopa in capsule samples
times (s). a 0 s, b 50 s, c 100 s, d 150 s, e 200 s, f 250 s.
To evaluate the practicability of the proposed method, we
measured levodopa in capsules by DPV. A portion of the powder
Table 1
equivalent to 10 mg of levodopa was accurately weighed, dissolved
Comparison of the analytical parameters for the determination of levodopa by using
different electrodes. into 10 mL of 50 mM pH 4.8 acetate buffer, and separated with a
centrifugal machine. Further dilution was also performed with
Electrode pH Linear range Detection limit References
(mM) (mM)
50 mM pH 4.8 acetate buffer to reach the calibration range of
levodopa. Quantitative analysis was performed by spiking a known
Gold screen-printed 3 99e1200 68 [32]
concentration of levodopa solution into samples. The recoveries of
electrode
Boron-doped diamond 3 2e1100 0.8 [33] the levodopa concentration ranged from 96.2% to 101% with RSD of
MgAI-LDH/ 2.8%e3.4%, The results of four spiked capsule samples are sum-
NiMn2O4/polyanilinc/GCE 7 0.1e1100 0.005 [20] marized in Table 2, confirming that the proposed method is reliable
Au-CNT/PGE 7 0.1e1150 0.05 [25] for levodopa determination in capsule samples.
poly(3-methyIthiophene)- 6 50e195 50 [34]
MW
CNT/GCE 4. Conclusion
pCFA-GCE 4.8 1e150 0.14 [19]
bare GCE 4.8 100e3000 40 This work In this work, the voltammetric behaviors of DA and levodopa
MgAl-LDH:MgAl layered double hydroxide. were compared. Both DA-quinone and levodopa-quinone could
CNT: carbon nanotube. produce the intramolecular cyclization reaction, and the rate of the
PGE: pyrolytic graphite electrode.
intramolecular cyclization reaction between them differed at the
MWCNT: multi-walled carbon nanotube.
pCFA: poly(caffeic acid).
same pH. A new pair of peaks occurred at negative potential in the
second cyclic scan, which corresponded to the product of cycliza-
tion. The cyclization of DA-quinone was completely suppressed,
aminochrome accumulated near the electrode surface, and the while that of levodopa-quinone occurred in pH 4.8 buffer. As a
height of peak 3 increased with the quiet time. In addition, Fig. 6 result, we developed a method for the distinct detection of levo-
reveals that aminochrome was stable in the pH 4.8 buffer solution. dopa with bare GCE in the presence of DA. This method has the
advantages of simple operation, strong anti-interference ability,
3.4. Calibration plot and interference study and good reproducibility.
Under quiet time of 5 s, DPV was utilized to detect the limit of CRediT authorship contribution statement
the proposed method in pH 4.8 acetate buffer. The extent of an
intramolecular reaction is independent of the substrate concen- Yaotian Wang: Formal analysis, Data curation, Writing e orig-
tration [28]. Therefore, the peak current (Ipc) of peak 3 had a linear inal draft, Designing and conducting experiments, data collection &
relationship with the concentration (C) of levodopa in the range of analysis. Haiyan Zhang: Supervision, Writing e review & editing.
0.1e3 mM. The linear regression equations can be expressed as Mingli Chen: Conceptualization, Supervision, Writing e review &
follows: Ipc (mA) ¼ 2.465 þ 4.305 C (mM) (R2 ¼ 0.999). The limit of editing.
detection (LOD) was calculated using the formula LOD ¼ 3 S/m
where S is the standard deviation of the intercept, and m is the Declaration of competing interest
slope of the regression line. The LOD was estimated to be about
0.04 mM. Table 1 shows a comparison of different electrodes for the The authors declare that they have no known competing
electrochemical detection of levodopa. The proposed method could financial interests or personal relationships that could have
provide an appropriate linear range for the determination of appeared to influence the work reported in this paper.
levodopa.
AA like DA may cause serious interference for the detection of Acknowledgements
levodopa because AA coexists with levodopa in human fluids at
high concentrations However, the existence of AA like DA had no This research did not receive any specific grant from funding
interference on the determination of levodopa. This phenomenon agencies in the public, commercial, or not-for-profit sectors.
5
Y. Wang, H. Zhang and M. Chen Analytica Chimica Acta 1157 (2021) 338379
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