Dopamine has the chemical formula (C8H11NO2).
Its chemical name is 4-(2-aminoethyl)benzene-1,2-diol and it is abbreviated
as DA (Benes, 2001). It belongs to the catecholamine family; it is a precursor
to epinephrine (adrenaline) and then norepinephrine (noradrenaline) in
the biosynthetic pathways for these neurotransmitters (Fig.
Dopamine is a phenethylamine naturally produced by the human body. In
the brain, dopamine functions as a neurotransmitter. Dopamine is also
a neurohormone released by the hypothalamus. Its main function as a hormone
is to inhibit the release of prolactin from the anterior lobe of the pituitary.
It is commonly associated with the pleasure system of the brain, providing
feelings of enjoyment and reinforcement to motivate a person proactively
to perform certain activities.
Structural formulas of dopamine (DA), N,N-bis-ethoxycarbonyl-1,10-diaza-4,7,13,16-tetraoxacyclo-octadecane
(DZCE) and potassium tetra-kis-[3,5-bis-(trifloro-methyl)-phenyl]
Deficiency in biosynthesis of dopamine
will result in Parkinson`s disease. In contrast, the production of more
dopamine is recorded for schizophrenic patients. Therefore, it is important
to follow up the dopamine level in both cases during, before and after
medications. Dopamine can be supplied as a medication that acts on the
sympathetic nervous system, producing effects such as increased heart
rate and blood pressure.
Different methods were used for the determination of dopamine. Flow Injection
Analysis (FIA) and HPLC with chemiluminescence detection were applied
for determination of DA by Nalewajko et al. (2007). Catecholamines
were determined by the spectrophotometric method based on the use of poly
(dimethylsiloxane) (PDMS) technology (Maminski et al., 2005). Abdulrahman
et al. (2005) reported a flow injection-spectrophotometric determination
of some catecholamine drugs via oxidative coupling reaction with p-toluidine
and sodium periodate. Nour El-Dien et al. (2005) described two
spectrophotometric methods for the determination of dopamine derivatives.
Glassy carbon electrodes were applied for the determination of dopamine.
A sensitive and selective electrochemical method for the determination
of dopamine (DA) was developed using a calix-4-arene crown-4 ether film
modified glassy carbon electrode (Lai et al., 2007). The Si-TiPH
bulk modified carbon paste electrode (Kooshki and Shams, 2007) and Nafion
coated carbon paste electrode (Alpat et al., 2005) was used for
the selective determination of dopamine (DA) in the presence of ascorbic
acid. Electrocatalytic oxidation and selective detection of dopamine at a 5,5-ditetradecyl-2-(2-trimethyl-ammonioethyl)-1,3-dioxane
bromide self-assembled bilayer membrane modified glassy carbon electrode
was introduced by Lin and Gong (2004). Hayashi et al. (2003) succeeded
in detecting dopamine (DA) in the presence of L-ascorbic acid using interdigitated
electrode array. Cheng et al. (2005) applied fiberion carbon-fiber
electrode for in vivo determination of concentration of cationic
Many researchers constructed amperometric sensors for dopamine. Cu-dipyridyl
complex was applied for preparing amperometric sensors for dopamine (Sotomayor
et al., 2002). Tu and Chen (2002) applied nano-disposable biosensors
for dopamine. Plant tissue containing oxidizing enzyme was applied to
build up biosensor for determination of dopamine and other catechol amines
(Mazzei et al., 1992).
Some previous researches were reported for the determination of DA using
ion-selective electrodes. Calix-3-arene derivative was applied for preparing
DA-selective electrode (Saijo et al., 2007). Júnior et
al. (2000) constructed flow injection analysis (FIA)-potentiometric
sensor for dopamine based on poly (ethylene-co-vinyl acetate)-Cu (II)
ion. Othman et al. (2004) prepared PVC-membrane electrodes based
on 12-crown-4-phosphotungestic and 12-crown-4-TPB ionophores for dopamine
determination. Dopamine was analysed either by an indirect method using
periodate selective electrode (Montenegro and Sales, 2000) or by direct
method using dopamine selective electrode (Lima and Montenegro, 1999)
based on tetra-chlorophenylborate. Earlier, Liu and Yu (1990) applied
crown ethers as neutral ionophores for preparing electrodes for a primary
In fruits, dopamine is the substrate for polyphenol oxidases (PPOs).
These are a family of enzymes responsible for browning of fresh fruits
and vegetables when they are cut or bruised. This helps to protect damaged
fruits and vegetables against growth of bacteria and fungi (Romphophak
et al., 2005).
In the present study, two highly selective sensors were introduced for
the determination of dopamine. A comparison between the properties of
the diaza crown and the previously reported crown based electrodes was
established. The new electrode allows the direct determination of dopamine
without pre-separation using toxic solvents or expensive instrumentation
like in HPLC. No need of liquid nitrogen, like in polarographic methods.
Ascorbic and uric acids were not interfering in dopamine determination
by this electrode which is recorded when using carbon paste electrodes.
An improvement of selectivity was found after adding synthesized diaza-18-crown-6
ether (DZCE) derivative to the potassium tetrakis-[3,5-bis-(trifloromethyl)
phenyl] borate (K-TFPB). Evaluation of selectivity properties was applied
by introducing and discussing the idea of Relative Selectivity Coefficient
RSC for the first time.
MATERIALS AND METHODS
Synthesis of the host molecule: DZCE was synthesized according
to the procedure described previously (Hodgkinson et al., 1979).
It depends on the addition of (5.84 g) of N,N-bis-ethoxycarboxycarbonyl-1,8-diamino-3,6-dioxaoctane
in dry dimethyl sulfoxide (DMSO) (75 cm3) dropwisely to a stirred
suspension of sodium hydride (1.12 g) in dry dimethyl sulfoxide (125 cm3).
After 3 h, triethylene glycol bis-p-sulfonate (10 g) in dry DMSO
was added to the formed solution and the mixture was sit for 3 days under
nitrogen. Then, HCl (2 M) was added and the mixture was extracted in CHCl3.
The organic extract was washed with water, dried in MgSO4 and
evaporated. The residual oil was purified by chromatography on silica. The macrocycle
DZCE was obtained as solid (m.p. 75-78°C). The structure of the obtained
compound was verified by elemental analysis and IR (found C, 52.9; H,
7.1; N, 8.4%; confirm C18H34N2O8;
vmax 1680 cm-1).
Reagents and materials: Materials used for the preparation of
electrodes were tetrahydrofuran (THF) (Merck) (after its distillation),
dodecyl phthalate (DDP) (Fluka), potassium tetra-kis-[3,5-bis-(trifloromethyl)
phenyl] borate (K-TFPB) (Fluka), high molecular weight poly (vinylchloride)
(PVC) (Fluka) and N,N-bis- ethoxycarbonyl-1,10-diaza-4,7,13,16-tetraoxacyclo-
octadecane (diaza-18-crown-6) (DZCE) (synthesized as aforementioned) were
used for the preparation of all membranes. Dopamine (4-(2-aminoethyl)benzene-1,2-diol)
(Sigma), ephedrineHCl (Sigma), adrenaline (Sigma), caffeine (Sigma), pilocarpine
hydrochloride (Sigma), atropine sulfate (Sigma); adrenaline (Sigma), ascorbic
acid, urea, glycine, arginine and sodium glutamate (Aldrich) were used.
Dopamine injection ampoules (40 mg/5 cm3) were purchased from
the local drug stores. Nitrate salts of inorganic cations (Na+,
K+, Li+, NH4+, Ca++,
Mg++ and Ba++) were purchased from (Fluka). De-ionized
water was operated through the whole work for the preparation of different
solutions and for rinsing the electrodes.
Instruments: The cell-EMF values were measured using a bench top model
Sension-4 (HACH, USA). The instrument was loaded to a computer system through
RS-232 connection and HACH-software. The same instrument was applied for pH-measurements.
The spectrophotometric measurements were carried by a UV-VIS-spectrophotometer
DR-4000 (HACH, USA), fitted with flow injection unit and loaded to a computer
system through RS-232 connection and HACH-software.
Electrode system and potentiometric measurements: One milligram
KTFPB or (1 mg KTFPB + 2 mg DZCE) were applied as sensor materials for
membranes I and II, respectively. The mentioned ionophores were mixed
with 60-67 mg DDP plasticizer and 30-31.5 mg PVC. The membrane was prepared
according to a procedure described before (Zareh et al., 2001).
The membrane discs were mounted on electrode Phillips bodies (type IS
561, Eindhoven, Netherlands) for electromotive force measurements. The
electrode was filled with an aqueous inner filling solution (KCl 0.01
M and DAHCl (0.01 M) solution). It was doped 24 h in 10-2 M,
before first use.
The potentiometric measurements were performed at room temperature (25°C)
in a Galvanic cell, which can be represented as the following:
Ag-AgCl/Li-acetate/Sample//Ion-selective membrane// Inner filling solution/Ag-AgCl
The outer compartment of the reference electrode was filled with lithium
acetate solution (0.1 M).
The potential was measured by immersing the electrodes with a reference
Calomel into water (50 cm3). Different aliquots of DAHCl (10-2
and 10-1 M) were added to cover a concentration range of DAH+
(10-7-10-2 M). The potential values were recorded
and plotted versus p[DAH+] using Microsoft Excel 2003. For
studying the pH-effect on the electrode potential, the pH was changed
using NaOH or HCl (0.1 M). The potential values were recorded at different
pH for the electrodes in 10-4, 10-3 and 10-2
M DAH+ solutions.
The selectivity coefficient values for several cations (Na+,
K+, Li+, NH4+, Ca++,
Mg++ and Ba++), aminoacids (glycine, arginine and
sodium glutamate) and pharmaceutical amines (ephedrineHCl, adrenaline,
caffeine, pilocarpine hydrochloride and atropine sulfate) are calculated
(Table 1) by the use of the Separate Solution Method
(SSM) (Guilbault et al., 1976). The emf of the interference solution
(0.01 M) and that for the same concentration of DAH+ solution
were measured. Then, the selectivity coefficient values of the electrodes
(Ki,j pot) were estimated for the different interferents
according to the equation:
log Kpot DAH+,
Jz+ = (Ej —
EDAH+) / S + [1-(ZDAH+/Zj)]
where, E represents the emf readings for the primary ion DAH+ and
the interfering ion (Jz+) and (S) is the observed slope for
the primary ion.
Injection ampoules of dopamine HCl (5 cm3) (products of EIPICO,
Egypt; Ebewe Pharma, Austria; or Pierre Fabre, France) were diluted to
50 mL solutions. The obtained solutions were transferred to the potentiometic
cell. Both DAH+-selective and the reference electrodes were
immersed into the solutions and the cell EMF was measured. The potential
readings of the sample solutions were compared to previously prepared
calibration graph under the same condition.
Twenty gram (fresh weight) of sliced banana pulp (two days ripened) were
transferred to a beaker containing 20 cm3 0.1 M HCl. The mixture
was homogenized in a blender. The homogenate was centrifuged for 20 min.
The supernatant solution was adjusted to pH 4 using K2CO3,
then brought up to 50 cm3. The solution was transferred to
the potentiometric cell and subjected for EMF-measurements using the proposed
electrode. The obtained values were compared to a calibration graph of
dopamine standard solutions treated typically like the measured samples.
For the spectrophotometric analysis, after extraction as aforementioned,
purification step was applied. It was performed by elution of 5 cm3
of the extract into a Dowex 50 X-8 column, which was washed with 20 mL
2 M HCl, 5 mL water, 10 cm3 1 N acetate-acetic buffer (pH 6)
and finally with 5 mL water. The dopamine was eluted by 6 mL 1 M HCl at
rate of 0.25 cm3 min-1, followed by 6 mL 2 M HCl.
The eluted dopamine was assayed according to procedures mentioned by Abdulrahman
et al. (2005). It is based on the oxidative coupling with p-toluidine
(0.008% w/v) and sodium periodate (0.4 mM) giving an orange dye with maximum
absorption at 480 nm. The obtained results were compared to a calibration
graph previously prepared for dopamine solutions (2-50 μg cm-3)
under the same conditions.
RESULTS AND DISCUSSION
Two electrodes with different membrane compositions were prepared. One
has a membrane containing KTFPB (type-I) and the other constitutes (KTFPB+DZCE)
(type-II). Both electrodes exhibit typical Nernstain slope (57.8 and 58.4
mV/decade). They work linearly down to 7.9x10-6 M DAH+.
The calibration graphs representing both electrodes are displayed in Fig.
2. The lower linear limit of the calibration graph for electrode II
is better than that of electrode I. This is because the ion-pair in electrode-II
contains a cavity, which helps the chelation of DAH+. The DZCE
works like the charge carrier that helps the ion-association with [TFPB]–.
The mechanistic equation that represents the exchange reaction at the
membrane-solution interface for electrode-I is written as below:
+ [TFPB]– ===== [ TFPB– DAH+]
= [ TFPB– DAH+] / [DAH+]
In case of electrode-II, the following equilibrium is expected:
K+ + DZCE ===== [(DZCE-K)+ TFPB–]
TFPB–] + DAH+ Cl– =====
[(DZCE-DAH)+ TFPB–] + KCl
= [(DZCE-DAH)+ TFPB–] / ([(DZCE-K)+
The dynamic response of both electrodes showed instantaneous and stable
potential readings. Figure 3 displays the obtained results.
The mV-pH curves for both electrodes (I and II) are shown in Fig.
4. The working pH-range is 3.5-8.3 for both electrodes for 10-2 M DAH+.
graphs for dopamine electrodes based on diazacrown ether derivative
of dopamine electrodes based on diazacrown ether derivative
pH on the potential of dopamine electrodes based on either TFPB and
(TFPB + DZCE) in 0.01 M dopamine solution
coefficient values (KDAH+, Jz+pot)
for both dopamine electrodes based on TFPB and diazacrown ether analogues
pH on the potential of dopamine electrode type-II based on K-TFPB
and DDP into 0.01 and 0.001 M dopamine solutions
Whenever 10-3 M DAH+ solution was measured, the plateau potential values
were between 2.9 and 9.1. This is a wide pH-range compared to the previous
electrodes, which are not working in alkaline medium (2.5-6 and 3.5-6). Figure 5 shows the mV-pH curves for the dopamine electrode-II
at 10-3 and 10-2 M concentrations. The formation
of the free base is the reason of the break in the basic part of the plateau.
This depends on the concentration of DAH+. In acidic medium,
the hydrogen ion interference is the reason of the curve break.
Selectivity and Relative Selectivity Coefficient (RSC): The selectivity
coefficient values (KDAH+jz+ pot) for
the common inorganic cations, amino acids, pharmaceutical amines were
calculated and recorded in Table 1. It is shown that
the values of the selectivity coefficients for the inorganic cations range
between 10-5 and 10-7. This shows a super selectivity
properties. Amino acids like glycine, arginine, glutamate showed values
of the selectivity coefficient of the order of 10-5-10-6.
This is because the presence of the Zwitter ion masked the amine group
from being part of chelation with the DZCE. This favors the discrimination
of DPH+ over them. In case of ascorbic acid, uric acid and
other pharmaceutical amines (pilocarpine, adrenaline, ephedrine and atropine),
the selectivity coefficient values were of the order of 10-2-10-3.
Although these values are higher than those for other cations, they are
still suitable for measuring dopamine in presence of them.
The relative selectivity coefficient Krel (RSC) is a parameter
that is introduced to evaluate the selectivity properties of electrodes
or sensors of different composition, but responding to the same ion. This
factor helps the mathematical comparison between these sensors or electrodes.
It can be calculated by using their selectivity coefficient values (Ki,j
pot). For example if I and II are two electrodes responding to the
same primary ion, the following equation can be applied to calculate RSC
for both electrodes towards each other, for one interferent:
= (Ki,j pot)I /(Ki,jpot)II
= (Ki,jpot)II / (Ki,jpot)I
If there are (n) interferents, the Average Relative Selectivity Coefficient
(ARSC) (K I:II)av, can be calculate as
= [Σ1→n (Krel)I:II-n]/n
= [Σ1→n (Krel)II:I-n]/n
As a general formula if there are (m) number of electrodes to be compared
with (X) electrode under study, a general formula (Zareh`s equation) can
= [(Krel-X:1)av + (Krel-X:2)av
+ (Krel-X:3)av + (Krel-X:4)av
+ .....+ (Krel-X:m)av]/(m)
where, (1, 2, 3, 4,....m): refers to the number of electrodes to be compared
with; (Krel-X)total : is the average of the relative
selectivity coefficient (TARSC) for X-electrode toward all electrodes.
It can be predicted that the smaller the (Krel-X)total-value,
the better the selectivity properties of an electrode.
The selectivity coefficient values are shown in Table 1.
The values (KDAH+, Jz+) for electrode
II is lower than those for electrode I. This means a better selectivity
for electrode II than electrode I is expected. Nevertheless, this is not
enough to discriminate between the selectivity of the two electrodes.
Therefore, evaluation of selectivity properties was conducted by applying
the aforementioned relative selectivity rules. Generally, it can be reported
that the selectivity coefficient values of electrode II has numerical
values less than that for electrode I by a factor 0.712. This factor is
the value of the relative selectivity coefficient RSC for electrode II
toward electrode I (Krel)II:I. For the inorganic
cations, the average (Krel)II:I is 0.723, while
for organic amines it is 0.703. Table 2, shows the obtained
results. This shows that the selectivity properties of electrode II is
better than that of electrode I toward these cations.
Coefficient (RSC) values for both I and II dopamine electrodes towards
This is caused —y
the association of the formed ion-pair by the host DZCE-molecule, which
led to more stability of the formed DAH+-complex.
Likewise, by using the RSC-concept the selectivity properties of electrode
I can be compared to the previously reported electrodes (pre1) and (pre2)
(Othman et al., 2004). Where, (pre1) and (pre2) refer to the previously
reported electrodes with crown ether-phosphotungestic acid (CE-PTA) and
crown ether-tetraphenyl-borate (CE-TPB) ion pairs, respectively. The total
average relative selectivity coefficients (TARSC) values for electrode
I are (Krel)I:pre1: 9.9x10-3 and (Krel)I:pre2:
5.0x10-3. By the same way electrode II is compared to previously
reported electrodes (pre1) or (pre2). It is found that (Krel)II:pre1
is 6.7x10-3 and (Krel)II:pre2 is 3.4x10-3.
This means that the present electrode exhibits better selectivity properties
than the previously reported electrodes. Table 3 shows
the calculated values.
The application of the proposed electrode for the determination of dopamine
in its pharmaceutical preparations was established. Samples of injection
solutions from different companies were subjected to the electrode analytical
procedures. Table 4 shows the obtained results. The
recovery showed values between 98.5 and 99.7%. The RSD values ranged between
1.32 and 1.64. The obtained results agreed with those obtained by applying
the previously reported spectrophotometric method (Abdulrahman et al.,
The electrode was applied for determination of dopamine in banana. The
results agreed with those obtained by the mentioned spectrophotometric
method. Table 5 summarizes the obtained results for
dopamine in banana pulp after 2 days ripening.
Coefficient (RSC) values for the present electrodes I and II toward
the previously reported (Othman et al., 2004) electrodes
(pre1) or (pre2) based on either (CE-PTA) or (CE-TPB), respectively
*Othman et al. (2004)
of dopamine in pharmaceutical samples using the proposed electrode
et al. (2005), **Four determinations
of dopamine in banana pulp samples after two days ripening using the
proposed electrode type-II
et al. (2005), **Relative standard deviation (4 determinations)
Application of originally formulated diazacrown ether derivative as a
new ionophore for preparing dopamine electrode showed successful results.
The selectivity and the sensitivity of electrodes based on the synthesized
diazacrown ethers were better than those for previously reported electrodes
based on the usual crown ethers.
The use of the new concept of the relative selectivity coefficient is
very useful in judgment between the different electrodes that are selective
to the same ion. It made the comparison more accurate and specific.