Journal of Electroanalytical Chemistry 521 (2002) 117 126

www.elsevier.com/locate/jelechem

Electrochemical detection of tricyclic antidepressant drugs by

HPLC using highly boron-doped diamond electrodes
T.A. Ivandini a, B.V. Sarada a, C. Terashima a, T.N. Rao a, D.A. Tryk a, H. Ishiguro b,
Y. Kubota b, A. Fujishima a,*
a
Department of Applied Chemistry, School of Engineering, The Uni6ersity of Tokyo, 7 -3 -1, Hongo, Bunkyo-ku, Tokyo 113 -8656, Japan
b
Department of Urology, School of Medicine, Yokohama City Uni6ersity, 3 -9 Fukuura, Kanazawa-ku, Yokohama 236 -8567, Japan

Received 13 August 2001; received in revised form 6 December 2001; accepted 12 December 2001

Abstract

Boron-doped diamond (BDD) electrodes have been examined for the electrochemical detection of six tricyclic antidepressant
drugs (TCAs): imipramine, desipramine, clomipramine, amitriptyline, nortriptyline, and doxepin. Cyclic voltammetry, flow
injection analysis (FIA) and HPLC with electrochemical detection have been used to study the oxidation reactions and to detect
the TCAs. At diamond electrodes, well-defined and highly reproducible voltammograms were obtained for all six drugs with a
signal to background (S/B) ratio about 24 times greater than those at glassy carbon electrodes. Diamond is the first electrode
material to show well-defined voltammograms for nortriptyline due to its wide potential window. In the FIA-mode, at an
operation potential of 0.85 V versus Ag  AgCl, diamond exhibited a background current of 7 nA with rapid stabilization (15 min)
conversely to the case of GC, which appeared to stabilize after 1 h, but again increased thereafter. The analytical peaks of HPLC
for the TCAs were well resolved. Linear calibration curves were linear over the ranges from 0.05 to 100 mM. The limits of
detection (S/N=3) were 3 nM for imipramine and desipramine, 0.5 nM for clomipramine, 163 nM amitriptyline, 1080 nM for
nortriptyline and 92 nM for doxepin. The electrodes have shown reproducible results over several days of analysis. This method
has been applied for the determination of imipramine and desipramine in plasma samples. The BDD surface was reproducible
with no adsorption of blood components during plasma analysis. This work shows the promising use of diamond as an
amperometric detector in HPLC, especially for TCA analysis. 2002 Published by Elsevier Science B.V.

Keywords: Tricyclic antidepressant drugs; Boron-doped diamond; HPLC; Plasma sample; Reproducibility

1. Introduction or no therapeutic effect. Furthermore, severe adverse

Tricyclic antidepressant drugs (TCAs) are one of the
largest groups of drugs for the treatment of psychiatric
disorders such as depression, mainly endogenous major
depressions. The function of these drugs is to block the
reuptake of the neurotransmitters norepinephrine and
serotonin in the central nervous system [1]. The chemi-
cal structures of these compounds are illustrated in
Scheme 1. For this group of drugs, distinct ranges of
optimal plasma concentration for therapy are required.
Lower concentrations are associated with sub-optimal
* Corresponding author. Tel.: + 81-3-3812-9276; fax: +81-3-3812-
6227.
E-mail address: akira-fu@fchem.chem.t.u-tokyo.ac.jp (A. Fu-
jishima).
effects and toxicity can appear at high concentrations.
Their toxicity results from their central and peripheral
anticholinergic effects, their peripheral a-adrenoceptor
blocking activity and their membrane-stabilizing activ-
ity, resulting in cardiovascular complication, convulsion
and coma. Therefore, the analysis of these compounds
is important for quality assurance in pharmaceutical
preparations and for obtaining optimum therapeutic
concentrations to minimize the risk of toxicity. The
therapeutic concentration range for most TCAs is ap-
proximately from 280 to 850 nM, while toxic effects can
occur when plasma concentrations exceed 1.4 mM [2,3].
Several methods are available to determine TCAs,
including radioimmunoassay, spectrofluorimetry, gas
chromatography and high performance liquid chro-
matography (HPLC) using UV absorbance, chemilu-

0022-0728/02/$ - see front matter 2002 Published by Elsevier Science B.V.

PII: S 0 0 2 2 - 0 7 2 8 ( 0 2 ) 0 0 6 6 6 - 6
118 T.A. I6andini et al. / Journal of Electroanalytical Chemistry 521 (2002) 117126
minescence or electrochemical detection [2 8]. For
therapeutic drug monitoring, where specificity is re-
quired, chromatographic methods are the most ade-
quate techniques. The advantages of HPLC for the
analysis of TCAs are its versatility and simplicity of
sample preparation. However, some trialkylamines and
related compounds are difficult to detect by use of UV
absorption, because they do not adsorb very well in the
UV visible region, since they have low molar absorp-
tivities [2]. The chemiluminescence method is promis-
ing, but the limit of detection was found to be relatively
high (500 nM) [2,46]. Electrochemical detection meth-
ods are very advantageous over other methods in terms
of simplicity, sensitivity, selectivity and cost. However,
these methods have not become as popular as other
methods due to certain unavoidable problems such as
electrode deactivation, with the necessity of frequent
pretreatment and other procedures to reactivate the
solid electrodes. Glassy carbon (GC), one of the widely
used electrodes for electrochemical detection, due to its
relatively wide potential window and low cost, is very
susceptible to contamination and fouling [9 12]. Al-
Scheme 1. Chemical structure of six tricyclic antidepressant drugs.
though few reports are available about the use of GC in
the HPLC-ED for the analysis of TCAs, the stability
aspects are not mentioned. Surmann and Peter, on the
other hand, have reported the influence of pretreatment
of carbon fiber electrodes on the analysis of bamipine
and imipramine [13]. Therefore, a stable electrode mate-
rial with sensitive detection capabilities is a prime re-
quirement for wider application of electrochemical
detectors. Recently emerging diamond materials meet
all these requirements to a great extent.
The use of highly boron-doped diamond (BDD) thin
films as electrode materials for several applications,
including electroanalysis [14 20], energy storage
devices [21], and electrosynthesis [22] has gained great
interest in the past few years due to the impressive
properties of such films. The superiority of diamond
thin films for electroanalysis results from their attrac-
tive properties, such as the wide electrochemical poten-
tial window in aqueous solution [23,24], very low
voltammetric background current ( 1 order of magni-
tude lower than GC) [25,26] inertness of the surface to
adsorption of reaction products [14], extreme electro-
 T.A. I6andini et al. / Journal of Electroanalytical Chemistry 521 (2002) 117126
chemical stability [21], and relative insensitivity to dis-
solved oxygen [27]. Several research groups including
our group have previously reported the uniqueness of
diamond for the detection of various environmentally,
biologically and clinically important compounds using
flow injection analysis or liquid chromatography, re-
sulting in sensitive and stable detection [15 20]. During
the use of other electrode materials such as metals, GC
and carbon paste, the sensitivity is decreased for the
detection of the compounds that oxidize at relatively
high potentials due to the interference of the oxygen
evolution reaction, or the requirement of frequent sur-
face pretreatment processes due to the strong adsorp-
tion of reactants and products on the electrode surface.
Recently, Rao et al. demonstrated the ascendancy of
diamond electrodes over GC for the detection of sulfa
drugs [20]. Therefore, BDD, with its inert surface prop-
erties could be a promising electrode for the electro-
chemical detection of TCAs using HPLC.
In the present study, we report the detection of six
TCAs, imipramine and its metabolite desipramine,
clomipramine, amitriptyline and its metabolite nor-
triptyline, and doxepin at neutral pH, by the use of
BDD electrodes for the electrooxidation. Cyclic voltam-
metry, flow injection analysis and HPLC with an am-
perometric detector were used for the detection.
Comparison experiments were performed using GC
electrodes.
2. Experimental
2.1. Preparation of highly boron-doped diamond
electrode
The highly BDD films were grown by the microwave-
assisted plasma chemical vapor deposition (CVD) tech-
nique with a commercial microwave plasma reactor
(ASTeX) Corp., Woburn, MA). Films were grown on
Si(100) substrates. The details of the preparation have
been described previously [14]. A mixture of acetone
and methanol in the ratio of 9:1 (v/v) was used as the
carbon source. B2O3, the boron source was dissolved in
the acetone+methanol mixture at a B/C molar ratio of
1:100. The boron concentration in the film prepared
119
confirmed by Raman spectroscopy. The results showed
the narrow, intense peak at 1332 cm 1, which is char-
acteristic of diamond and reflects a high degree of
crystallinity. In addition, a broad peak centered at
approximately 1200 cm 1 was observed, which is usu-
ally attributed to either amorphous diamond or ex-
tremely small diamond crystallites [21].
2.2. Voltammetric in6estigation
Cyclic voltammetric measurements were carried out
in a single compartment cell with a saturated calomel
electrode (SCE) as the reference electrode and a plat-
inum wire as the counter electrode. The BDD working
electrode along with the conducting Si substrate or GC
electrode was pressed against the bottom of the glass
cell by a Viton O-ring. The electrical contact for BDD
was made through the backside of the scratched Si
substrate by contacting the brass current collecting
back plate. Before use as a working electrode, BDD
film was pretreated by ultrasonication in 2-propanol for
about 10 min followed by rinsing with high purity-wa-
ter. The purpose of this pretreatment is to remove the
organic impurities that may have remained or formed
during the deposition of diamond in the CVD chamber.
The GC electrode (GC-20 plate or GC-20 rod, Tokai
Carbon Co., Ltd) was pretreated by polishing with
diamond paste (Fujimi), followed by ultrasonication in
2-propanol before the experiment. The supporting elec-
trolyte was a mixture of 0.05 M KH2PO4 + 0.05 M
K2HPO4 (KH2PO4/K2HPO4, pH 6.9 90.1). The geo-
metric area of the working electrode was estimated to
be 0.09 cm2.
2.3. Flow injection analysis
The FIA system used in the present study, consisted
of a micro-LC pump (Bioanalytical System, LC-100),
an injector (Rheodyne) with a 20-ml injection loop, an
amperometric detector (Bioanalytical Systems, LC-4C),
and an XY recorder (Graphtec, WX400). The flow
rate set for the pump was 1 ml min 1 and was confi-
rmed before every experiment by measuring the volume
of the buffer collected at the outlet for 10 min. The
under these conditions was ca. 1.5 1021 cm 3 as
estimated by Notsu et al., based on nuclear reaction
measurements [11B(p,a)8Be], carried out by means of
1-meV proton bombardment and subsequent compari-
son of the a-spectrum in the 6 8 MeV region with a
BN standard [28]. High purity hydrogen was used as
the carrier gas. The bubbling of the acetone+
methanol +B2O3 solution was carried out at 25 C.
The deposition of the film was carried out at a mi-
crowave power of 5 kW. A film thickness of  40 mm
was achieved after 10 h deposition. The film quality was
wall-jet type flow cell consisted of the Ag  AgCl  1 M
LiCl reference electrode and a stainless steel tube as the
counter electrode, which also served as the tube for the
solution outlet. A 0.5-mm-thick silicon rubber gasket
was used as a spacer in the cell. The geometric area was
estimated to be 0.64 cm2. The cell volume was esti-
mated to be 24 ml by assuming a 25% compression of
the gasket. Phosphate buffer (KH2PO4 + K2HPO4, pH
6.99 0.1) was used as the mobile phase. The analyte
solutions were prepared with the same buffer. The
injection volume of the analyte was 20 ml.
120 T.A. I6andini et al. / Journal of Electroanalytical Chemistry 521 (2002) 117126
Hydrodynamic voltammograms were obtained for
each compound prior to amperometric determination
and the detection potential was chosen in the limiting
current range of the voltammogram.
2.4. HPLC analysis
The HPLC system consisted of a reversed-phase C18
column (Inertsil ODS-3 1504.6 mm ID; particle size,
5 mm) connected to the FIA system. The chro-
matograms were obtained at an applied potential 0.85
cyclic voltammetric behavior of the TCAs. At the BDD
electrode, the oxidation of imipramine occurred at
slightly higher potentials ( 0.844 V vs. SCE) than that
at the GC electrode ( 0.800 V vs. SCE). It is estab-
lished that diamond exhibits higher overpotentials for
inner sphere redox reactions in comparison to GC, with
the oxygen evolution and hydrogen evolution reactions
being the best examples. At both the electrodes, the
voltammogram exhibited similar features, i.e. irre-
versible oxidation peaks. However, for BDD, the low
background current (0.19 nA at 0.844 V vs. SCE) at the
V versus Ag  AgCl  1 M LiCl for the imipramine/de- oxidation potentials, compared to that at GC (1.39 nA
sipramine couple, at 0.93 versus Ag  AgCl for at 0.8 V vs. SCE), resulted in a large S/B ratio. Table 1
clomipramine and at 1.3 V versus Ag  AgCl for the presents a comparison of data for S/B ratios obtained
amitriptyline/nortriptyline couple and doxepin. The from the cyclic voltammetric data for the oxidation of
temperature was maintained at 26 C. All the experi-
ments were performed with a mixture of acetonitrile
and phosphate buffer 0.05 M (KH2PO4/K2HPO4 pH
6.99 0.1) in the ratio of 375:625 (v/v) as the mobile
phase for the detection of all TCAs, except for
clomipramine (50:50).

2.5. Sample preparation

A blood sample was collected from a male patient

(16 years old, 60 kg), 10 h after intake of an oral dose
of 10 mg imipramine (Tofranil, sugar-coated commer-
cial tablet). This sample was centrifuged for 5 min at
3000g and stored at 20 C until analysis. A 200-ml
portion of 20% HClO4 was added into 200 ml plasma
sample and vortex mixed for 5 min and then cen-
trifuged for 5 min. The supernatant was then neutral-
ized using NaOH. The final solutions were filtered using
a 0.4 mm pore filter and injected directly into the HPLC
column. Dilution was by a factor of 4.

2.6. Materials

Imipramine, desipramine, clomipramine, amitri-
ptyline, nortriptyline, doxepin, KH2PO4, K2HPO4,
HClO4, NaOH (Wako Chemical Co.) and acetonitrile
(Nacalai Tesque) were used without further purifica-
tion. All of these solutions were prepared using ultra-
pure (18 MV) water.
3. Results and discussion
Fig. 1. Cyclic voltammograms for 10 mM imipramine in 0.1 M
phosphate buffer (pH 6.9) at: (A) at a BDD electrode (B) a GC
electrode. Dashed lines show the voltammograms after stirring the
solution. The sweep rate was 100 mV s 1.
Table 1
Comparison of S/B ratio obtained from the cyclic voltammetry data
for the oxidation of TCAs in 0.1 M phosphate buffer (pH 6.9) for
BDD and GC electrode
BDD GC

3.1. Voltammetric studies Ep/V vs. SCE S/B Ep/V vs. SCE S/B

Fig. 1 shows the cyclic voltammograms obtained at a Imipramine 0.844 52.31 0.800 12.59
potential sweep rate of 100 mV s 1 for a deaerated Desipramine 0.833 55.84 0.833 33.00
Clomipramine 0.936 40.43 0.877 11.85
solution of 10 mM imipramine in 0.1 M phosphate
Amitriptyline 1.333 19.17 0.979 10.70
buffer (pH 6.9) together with the corresponding back- Nortriptyline 1.350 10.23 Undetectable
ground voltammogram, at BDD (Fig. 1A) and GC Doxepin 1.300 21.86 0.895 8.09
(Fig. 1B). In fact, deaeration has little effect on the
 T.A. I6andini et al. / Journal of Electroanalytical Chemistry 521 (2002) 117126
100 mM concentrations of all five TCAs in 0.1 M
phosphate buffer at BDD and GC. The results show
that the S/B ratios were about 2 4 times higher for
BDD compared to GC. The relatively low background
current for diamond was attributed to the predominant
hydrogen termination of the surface as well as the
compact nonporous surface [16,20]. Films used in this
study are predominantly hydrogen terminated, as they
were prepared in hydrogen plasma. Such films showed
an O/C ratio of  0.03. Earlier, Yagi et al. from our
group reported that XPS results of as-deposited BDD
showed only a sharp C 1s peak and no discernable O 1s
peak, which is typical for the H-terminated surface [29].
However, although the presence of oxygen functional
groups on the GC surface is believed to be partly
responsible for its high background current [9], our
previous studies have shown that surface oxygen does
not play a major role in the case of diamond in
determining the background current. An increase of
O/C ratio from about 0.03 to 0.18 changed its back-
ground to about 3 5 times, which was still much lower
than that of glassy carbon [29].
Another important observation is the recovery of the
voltammetric peak at a BDD electrode. The oxidation
curve of imipramine at BDD could be recovered by
stirring the solution for 2 min after the second cycle
(Fig. 1A). In the case of GC, the cyclic voltammogram
could not be retraced even after vigorous stirring, indi-
cating partial deactivation of the electrode surface
probably due to the adsorption of oxidation products
(Fig. 1B). The oxidation peak current at GC was about
4 times larger than that obtained at BDD. This may be
due to the adsorption of the compound on the electrode
Scheme 2. Electrooxidation mechanism of imipramine.
121
surface. At GC, strong adsorption on the surface gener-
ally provides high sensitivity for the detection of TCAs,
but the adsorbed reaction products form insulating
layers on the surface resulting in electrode fouling [20].
The shoulder peak observed at higher potentials for
both the electrodes may be due to the oxidation of a
reaction product. Similar voltammograms were ob-
tained for desipramine, a metabolite of imipramine. In
the case of clomipramine, a higher potential (V0.94
V vs. SCE) was needed to oxidize the compound, since
clomipramine contains highly electronegative substi-
tuted chlorine that stabilizes the compound and makes
oxidation more difficult.
At the BDD electrode the peak currents increased
linearly with the square root of the sweep rate (w 1/2)
within the range of 5300 mV s 1 (r 2  0.9996) with a
zero intercept. The linearity suggested that the current
is limited by diffusion control in the interfacial area of
the electrode and the adsorption steps and specific
surface interaction can be neglected.
The electrochemical oxidation of imipramine, de-
sipramine and clomipramine are believed to occur at
the nitrogen atom in cyclohexane involving a two-elec-
tron one-proton mechanism, in analogy to that for
methyliminobibenzyl [30,31], as illustrated in Scheme 2.
The dimer is formed, which is more easily oxidized than
the monomer. The redox-peak pair that appeared at
 0.037 V after the first cycle may be due to the dimer,
since after the first oxidation, the dimer is formed and
is available near the electrode surface [32]. A simple
experiment was performed to investigate whether this
peak was due to the adsorption of reaction products on
the electrode surface. After stirring the solution for 2
122 T.A. I6andini et al. / Journal of Electroanalytical Chemistry 521 (2002) 117126

min, the electrodes were rinsed and the background

voltammograms were recorded for both BDD and GC.
At BDD, the background voltammogram was identical
with that of the fresh electrode. However, at a GC
electrode, the peak pair at 0.037 V versus Ag  AgCl
remained indicating a strong adsorption of the reaction
products on the electrode surface (Fig. 2). The large
peak currents and change in the background voltam-
mogram indicate the failure of GC for the clinical
detection of TCAs. Although dimer formation occurs
at the BDD electrode, it is not permanently adsorbed
on its surface. Surmann and Peter discussed in detail
the effect of electrochemical pretreatment of carbon
fibers for the detection of TCAs [13]. Another remote
possibility is the chemical oxidation of this dimer by the
monomer radical [33] to produce the unoxidized
azepine. The shoulder peak in the cyclic voltammogram
in Fig. 1 is possibly due to this azepine, although, at the
moment, we do not have evidence for this.
This mechanism suggests a pH dependence of the
overall process. Indeed, decreasing the pH of the solu-
tion resulted in a shift of the TCA oxidation peaks Fig. 2. Comparison of background current before and after
toward more positive potentials. Fig. 3 shows the varia- imipramine oxidation at: (A) a BDD electrode and (B) a GC elec-
tion of peak potentials for TCA oxidation at a BDD trode. Dashed lines show the voltammograms after imipramine oxi-
electrode as a function of pH. It can be seen that the dation. The sweep rate was 100 mV s 1.
peak potentials of imipramine, desipramine and
clomipramine shift linearly toward more positive poten-
tials with slopes (dE/dpH) ranging between 23 and 28
mV which is suitable for a mechanism involving two
electrons and one proton.
Cyclic voltammograms were also observed for
amitriptyline, nortriptyline, and doxepin, other types of
TCAs, which differ from imipramine, desipramine and
clomipramine because there is no nitrogen atom in the
cycloheptene ring. At BDD, the oxidation peaks of
amitriptyline, nortriptyline and doxepin appeared at
higher potential of about 1.3 V vs. SCE, higher than at
GC (0.9 V vs. SCE). However, at GC, the electrode
surface was easily fouled and the cyclic voltammograms
could not be retraced even after vigorous stirring. An
important observation was that the GC electrode gave
an ill-defined peak for nortriptyline as shown in Fig.
4b, due to the interference of the oxygen evolution
reaction together with oxidation of the GC electrode
surface itself.
Nortriptyline and doxepin, which have no ring nitro-
gen atoms, were previously reported to be electrochem-
ically inactive [33]. Although doxepin contains an
oxygen heteroatom, it does not present attackable lone
pairs. Then, Turk et al. found that the electroactivity is Fig. 3. Dependence of peak potential on pH of the solution for the
achievable by modifying the GC electrode with electro- oxidation of TCAs.
polymers such as polycarbazole or polythiophene. This
is due to the presence of electron-rich sulfur or nitrogen
atom on the polymer surface, which facilitates the amitriptyline and doxepin occurred with well-defined
formation of a cation radical [34]. However, at both oxidation peaks. The advantage of BDD, which is due
BDD and GC electrodes the electrooxidation of to its wide potential window, is that a well-defined peak
 T.A. I6andini et al. / Journal of Electroanalytical Chemistry 521 (2002) 117126 123

0.1 M phosphate buffer were sigmoidal, as shown in

Fig. 5. The potentials for the oxidation currents are in
the expected order while the imipramine derivatives
containing ring nitrogen oxidized at lower potentials
and doxepin, nortriptyline and amitriptyline oxidized at
higher potentials, as expected. The amperometric re-
sponse obtained for a BDD electrode for 20 ml injec-
tions of 100 mM imipramine gives reproducible peaks,
with a peak variability of  3% (n=18) suggesting no
adsorption of oxidation products on the electrode sur-
face. Reproducible peaks were obtained at BDD for
concentrations as low as 10 nM. (Fig. 6A), which could
be clearly distinguished from the peaks for buffer injec-

Fig. 4. Cyclic voltammograms of 100 mM nortriptyline in 0.1 M

phosphate buffer (pH 6.9) at: (a) BDD and (b) GC electrodes. The
sweep rate was 100 mV s 1.

at high potential occurred for nortriptyline, even

though the S/B ratio was quite small compared to the
other types of TCAs. Fig. 5. Hydrodynamic voltammogram for 20-ml injections of 10 mM
TCAs in 0.1 M phosphate buffer (pH 6.9) at BDD electrodes.
Due to the high oxidation potential of amines, no
reports were available on a voltammetric investigation
of these drugs on solid electrodes. However, Greenway
and Dolman suggested that the electrooxidation of
amitriptyline, nortriptyline and doxepin takes place at
the nitrogen atom in the alkylamine, resulting in the
formation of a cation radical, followed by deprotona-
tion [2]. The pH dependence of the voltammetric peak
current (Fig. 3) showed the same behavior for
amitriptyline, nortriptyline and doxepin, i.e. the peak
potentials shift linearly to more positive potentials with
increasing pH with slopes (dE/dpH) ranging between
58 and 67 mV, which suggests the participation of equal
numbers of protons and electrons in the oxidation
reaction. Detailed mechanistic studies for these com-
pounds at diamond electrodes will be conducted in the
future. The oxidation of amitriptyline and doxepin
occurred at lower potentials than that of nortriptyline,
since the former contain tertiary amine functional
groups, which are easier to oxidize [4].

3.2. Flow injection analysis Fig. 6. FIA-ED results for 20-ml injections of: (A) 10 nM imipramine
at a BDD electrode and (B) 10 nM imipramine at a GC electrode.
Hydrodynamic voltammograms obtained at the dia- The mobile phase was 0.1 M phosphate buffer pH 6.9. The flow rate
mond electrode for 20-ml injections of 10 mM TCAs in was 1 ml min 1.
124 T.A. I6andini et al. / Journal of Electroanalytical Chemistry 521 (2002) 117126

tions. In the case of GC electrodes, the response for
100 mM decreased to 72% after 18 injections. For 100
nM imipramine, the peak variability was  10%. Am-
perometric peaks obtained for 10 nM imipramine
were highly irregular due to the large noise in the
background current (Fig. 6B). The BDD electrode
showed a smaller degree of noise in the baseline, with
a low background current, compared to those at GC
of the electrode surface itself. Furthermore, at the end
of the experiment, a visible thin film formation on the
GC surface was observed, which is probably due to
the adsorption of contaminants from the buffer. GC
is prone to such contaminant deposition [12]. No visi-
ble change was observed on the diamond electrode.
Similar observations were made by Sarada et al. for
GC at 1.28 V and for BDD at 1.52 V. versus
electrodes. Similar results were obtained for other
TCAs. A linear dynamic range is obtained from 0.01
to 100 mM (r 2 =0.995), with an experimental detec-
tion limit of 0.01 mM for all TCAs, and 0.1 mM for
nortriptyline as an exception. At GC, the peak cur-
rent was saturated at high concentrations and the ex-
perimental limit of detection was 0.1 mM for all
TCAs, which is one order higher than those achieved
at BDD. Nortriptyline was not detectable at GC.
3.3. Chromatographic detection
Prior to the chromatographic studies of the TCAs,
the stabilities of electrodes were examined and com-
pared over a period of 10 h, for two types of com-
mercial GC electrodes, and a diamond thin film at
Ag  AgCl [35]. The noise in the background current
after 10 h of experimentation was about one order of
magnitude higher compared to that for BDD. The
low noise at BDD shows that very low detection lim-
its can be obtained. For the clinical applications, dur-
ing therapeutic monitoring, the stability of the
electrode surface is highly important and BDD sa-
tisfies this requirement. Also, the background current
at the BDD electrode was about 3 5 times less than
that for the GC electrodes. This enables the detection
of TCAs at very low concentrations.
Since desipramine is a metabolite of imipramine
and nortriptyline is a metabolite of amitriptyline, the
chromatograms were obtained for imipramine and de-
sipramine simultaneously (Fig. 8A) and similarly, for
the detection potentials, i.e. 800 mV versus Ag  AgCl amitriptyline and nortriptyline (Fig. 8B). Chro-
for GC and 850 mV versus Ag  AgCl for BDD (Fig.
7). At BDD, the background current stabilized within
20 min after switching to the detection potential and
the current was very stable during the 10 h of experi-
mentation. However, at both types of GC, longer
times (\ 60 min) were required to obtain consider-
able stability and there was a continuous change in
the current during the 10 h of experiment. The con-
tinuous change in the current with time was either
due to the adsorption of impurities or the oxidation
matograms for clomipramine and doxepin were ob-
tained separately (Fig. 8C and D). The mobile phase
was acetonitrile+ phosphate buffer at a ratio of
375:625 for all TCAs except for clomipramine (50:50),
since the Cl atom in its structure makes for high
polarity. The elution times for desipramine and
imipramine were 7.5 and 16.3 min, respectively, and
for clomipramine it was 10.55 min. Nortriptyline and
amitriptyline eluted at 7.66 and 18.5 min, respectively,
and doxepin eluted at 10.39 min.
TCA calibration curves obtained for the chro-
matograms were linear over the range 1000.05 mM.
The limit of detection for imipramine and de-
sipramine was 3 nM (S/N = 3). The lowest limit of
detection that can be achieved is 0.5 nM for
clomipramine. The linear calibration equation and
limits of detection (S/N = 3) are shown in Table 2.
BDD electrodes showed good linearity and reproduci-
bility for the detection of TCAs. These detection lim-
its were  5 times less than those reported previously
with an electrochemical detector [36]. The results were
reproducible from film to film, and with the same
film, reproducible peaks were obtained for the 3
months over which the experiments were performed.
Ultrasonication in 2-propanol for 10 min was suffi-
cient for cleaning the electrode surface when it was
used after a long interval. No mechanical or electro-
Fig. 7. Current vs. time profiles for BDD (at 0.85 V vs. Ag  AgCl),
GC20 Tokai and GC GL Tokai (at 0.80 V vs. Ag  AgCl). The mobile
chemical pretreatment was required before each set of
phase was acetonitrile +0.5 M phosphate buffer (pH 6.9) = 37.5:62.5. experiments, whereas GC required pretreatments in-
The flow rate was 1 ml min 1. cluding mechanical and electrochemical treatments.
 T.A. I6andini et al. / Journal of Electroanalytical Chemistry 521 (2002) 117126
Fig. 8. Chromatograms obtained from 10 mM concentration of: (A)
imipramine and desipramine, (B) clomipramine, (C) amitriptyline and
nortriptyline, and (D) doxepin, using an Inertsil ODS-3 column. The
mobile phase was acetonitrile + 0.5 M phosphate buffer (pH 6.9) =
37.5:62.5 except for (B) the mobile phase is acetonitrile +0.5 M
phosphate buffer (pH) = 50:50. The flow rate was 1 ml min 1, the
oven temperature was 26 C and the detector was ECD/BDD. The
125
Fig. 9. Chromatogram of plasma sample obtained from a patient
treated with 10 mg imipramine (Tofranil). Dilution factor is 4. Other
conditions are the same as Fig. 8A.
several proteins during the extraction process. Fig. 9
shows the chromatogram obtained for supernatant of
the extracted blood sample obtained from a patient
who was being treated for depression. After intake of
an oral dose of 10 mg imipramine, as already men-
tioned above, imipramine is metabolized to de-
sipramine. Therefore, peaks for imipramine and
desipramine were observed in the chromatogram. From
the currents obtained for amperometric peaks of HPLC
for the diluted blood sample, the average amounts of
desipramine and imipramine were found to be 0.126
mM (38.2 mg l 1) and 0.192 mM (60.8 mg l 1), respec-
tively (n= 3).
The recovery test was done using a blank plasma
sample spiked with 1.0 nmol (1.01 and 1.06 mg l 1) of
desipramine and imipramine, respectively, in a blank
operation potential, + 0.85 V vs. Ag  AgCl for (A), + 0.93 V vs.
Ag  AgCl for (B) and + 1.30 V vs. Ag  AgCl for (C) and (D).
3.4. Detection of TCAs in human-blood sample
BDD electrodes are most advantageous for the anal-
ysis of biological fluids, such as blood due to the lack of
adsorption of their components on the electrode sur-
face. At GC, the electrode surface is deactivated due to
adsorption of the components even after removal of
sample. The average recoveries of desipramine and
imipramine were 92.3 and 90.8%, respectively (n=3) as
shown in Table 3. The reproducibility is good with a
peak variability of  3%.
The reproducibility of the BDD electrode surface
after plasma analysis was confirmed by comparing the
peak currents obtained for a 1.0 mM standard solution
concentration of desipramine and imipramine before
and after the analysis. Good reproducibility was ob-
tained with peak variabilities of 0.35 and 3.27% (n=4)

Table 2
Calibration linear data for TCAs by using HPLC

Calibration equation Regression coefficients LOD/nM based on S/N=3

Imipramine y= 0.768+12.980x 0.9976 3

Desipramine y= 0.345+7.405x 0.9942 3
Clomipramine y= 1.935+7.735x 0.9988 0.5
Amitriptyline y= 5.098+5.425x 0.9996 163
Nortriptyline y= 0.922+1.073x 0.9994 1080
Doxepin y= 4.886+9.347x 0.9968 92
126 T.A. I6andini et al. / Journal of Electroanalytical Chemistry 521 (2002) 117126
Table 3
Recoveries from blank plasma spiked with 1.0 nmol of each de-
sipramine and imipramine (n= 3)
Compound Recovery
Amount Percentage/% SD/nmol CV/%
found/nmol
Imipramine 0.9089 90.89 0.0209 2.29
Desipramine 0.9283 92.83 0.0252 2.71
for desipramine and imipramine, respectively. The data
indicated that there was no fouling at the BDD electrode
after plasma analysis. The result shows that this method
can be used for the rapid analysis of imipramine and
desipramine in blood samples.
4. Conclusions
Conductive boron-doped diamond electrodes exhib-
ited an excellent performance for the electrochemical
detection of TCAs. Cyclic voltammetric studies show the
superiority of diamond over GC in terms of reproduci-
bility and sensitivity (S/B ratio). Diamond is the first
material to yield well-defined cyclic voltammograms for
nortriptyline on the unmodified surface due to its wide
potential window. FIA and HPLC results indicate that
the TCAs examined in this study are detected ampero-
metrically with high sensitivity and reproducibility. The
limit of detection (S/N =3) ranges from 0.5 nM for
clomipramine to 1.08 mM for nortriptyline. The back-
ground current stability test for diamond indicated faster
current stabilization after switching to the detection
potential (15 min) with a lower background current
(7 nA) in comparison to GC electrodes, which exhibited
a larger variation in background current during 10 h of
experimentation. The result obtained for the detection of
imipramine and desipramine in plasma samples showed
that the use of a BDD electrode with HPLC for the
detection of TCAs is rapid and reproducible with no
adsorption of the electroactive component of blood on
the electrode surface. These results demonstrated the
possibility of the practical utility of diamond in amper-
ometric detection coupled with FIA and HPLC systems
especially for TCAs.
Acknowledgements
This research was supported by the Japan Society for
the Promotion of Science (JSPS), Research for the Future
Program, Exploratory Research on Novel Artificial
Materials and Substances for Next Generation Indus-
tries.
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