Sensors and Actuators B 205 (2014) 215218

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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Short Communication

Flow-injection amperometric determination of yohimbine alkaloid in

dietary supplements using a boron-doped diamond electrode
a,

Lubomr Svorc
, Kurt Kalcher b
a
Institute of Analytical Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Radlinskho 9, Bratislava
SK-812 37, Slovak Republic
b
Institute of Chemistry, Department of Analytical Chemistry, Karl-Franzens University, Universittsplatz 1, Graz A-8010, Austria

a r t i c l e

i n f o

Article history:
Received 29 April 2014
Received in revised form 31 July 2014
Accepted 25 August 2014
Available online 3 September 2014
Keywords:
Yohimbine
Boron-doped diamond electrode
Flow-injection analysis
Detection limit
Recovery

a b s t r a c t
A simple, rapid and sensitive ow-injection method for the amperometric determination of the alkaloid
yohimbine using a boron-doped diamond electrode has been developed. Best conditions for owinjection analysis were a detection potential of +1.20 V (vs. Ag/AgCl) and a ow rate of 1.4 mL min1
with an injection volume of 100 L in BrittonRobinson buffer solution at pH 7 as a carrier. The current
response of yohimbine was proportional in the concentration range from 0.3 to 100 M with two linear segments (0.310 and 10100 M) and a low detection limit of 0.15 M (0.053 mg L1 ) as well as
good repeatability (relative standard deviation of 4.5% for n = 10) were achieved. The sampling frequency
was calculated about 70 injections h1 . The practical analytical usefulness of the developed procedure
was successfully demonstrated in the determination of yohimbine in dietary supplements with good
recoveries (9498% and 9295%) for Pausinystalia yohimbe and Rauvola serpentina, respectively.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Yohimbine
(17-hydroxy-yohimban-16-carboxylic
acid
methyl ester, YOH) is a well-known indole alkaloid primarily
acting as monoamine oxidase enzyme inhibitor [1]. It is antagonist
of 2 -receptors which increase brain noradrenaline cell ring
and release [2]. The side effects of YOH are usually accompanied
with anxiety, headache and increased urinary output [3]. The
bark of Pausinystalia yohimbe and Rauvola serpentina contains
YOH as main species used for clinical and traditional treatment
of sexual dysfunction and enhancement of sexual satisfaction
[4].
A survey in scientic literature reveals some papers dealing
with various analytical methods for the detection and quantication of YOH. Most of them include separation methods such as gas
chromatography with mass spectrometry [5], high-performance
liquid chromatography (HPLC) with UV detection [6] and mass
spectrometry [7] as well as capillary electrophoresis [8]. These
methods offer sensitive and selective determination of YOH,
however, they require long analysis time and time-consuming sample pretreatment processes as well as highly sophisticated and

Corresponding author. Tel.: +421 0 2 59325302; fax: +421 0 2 59325590.

E-mail address: lubomir.svorc@stuba.sk (L. Svorc).

http://dx.doi.org/10.1016/j.snb.2014.08.071
0925-4005/ 2014 Elsevier B.V. All rights reserved.

expensive instrumentation [9]. Prior to separation in chromatographic column, the general procedure for extraction and isolation
of indole alkaloids including YOH, reserpine, ajmalicine etc. usually
consists of following steps: maceration in methanol, evaporation,
dissolution in HCl, ltration, basication to the neutral pH, extraction in chloroform, evaporation and nally dissolution in methanol
with subsequent ltration [10]. It was found that extraction and
isolation procedure for these alkaloids may be modied depending on the purpose of the particular work [11,12]. The separation
methodology with this procedure enables the studied indole alkaloids to be sensitively and selectively quantied. However, despite
signicant performance of separation methods, the development of
novel, inexpensive, simple and rapid analytical alternative methods for the sensitive determination of YOH and other alkaloids in
various matrices is still of great interest.
Electroanalytical techniques have conrmed to be excellent
alternatives for the determination of various compounds, since
they are simple, cheap and require relatively short analysis time.
Moreover, they yield useful information about the kinetics and
charge transfer mechanisms of electrode reaction. Regarding
YOH, there is a short report available on its basic voltammetric
characterization using platinum and gold rotating disk electrodes
[13]. In the aforementioned study, YOH rendered an oxidation
peak at a potential higher than +1 V vs. SCE in 0.1 M sulphuric acid
with signicant adsorption contribution not enabling so sensitive

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L. S vorc, K. Kalcher / Sensors and Actuators B 205 (2014) 215218

determination of YOH (also with higher background current). As

to the combination of separation and electrochemical techniques,
an assay for the determination of YOH in human plasma utilizing
HPLC with glassy (vitreous) carbon electrode as an amperometric
detector was described by Goldberg et al. [14]. The low detection
limit for YOH of 0.025 M was obtained by application of the
oxidation potential of +0.95 V vs. Ag/AgCl electrode and reserpiline
as an internal standard. Hariharan et al. [15] developed a simple
and very sensitive HPLC method with coulometric detection also
with reserpiline as internal standard. The optimum oxidation
potential for YOH was found to be +0.80 V vs. Ag/AgCl electrode
with very low detection limit of 0.51 nM. On the basis of these
results, the combination of HPLC technique with amperometric
and coulometric detection appeared to be a sensitive platform for
quantication of YOH in biological samples and could be used for
pharmacokinetic study of indole alkaloids in humans.
Boron-doped diamond (BDD) has recently attracted a great
attention as modern electrode material opening new possibilities
in electroanalysis due to its superior properties such as wide potential range, low background current, high sensitivity and long-term
stability as well as negligible adsorption of polar species [16]. It
represents an effective alternative to conventional electrode materials such as platinum, graphite, glassy carbon and carbon paste.
Some data regarding the electrochemical behavior of YOH on BDD
electrode have been reported by Swain and co-workers [17]. Our
research group has recently published the paper regarding the
voltammetric determination of YOH by differential pulse voltammetry using BDD electrode [18]. In this study, two irreversible oxidation peaks, a distinct (quantication) peak at +0.80 V assigned to
the oxidation of the hydroxyl group of YOH including the loss of two
electrons and two protons to give a ring ketone and a second poorly
dened peak at +1.65 V associated with the oxidative deprotonization of the carbon at position 6, were observed in BrittonRobinson
buffer solution at pH 7. The linear concentration range was
appeared to be from 0.25 to 90.9 M with the detection limit of
0.13 M. The practical usefulness of this method in batch arrangement was demonstrated by the assessment of the total content of
YOH in extracts of the primary bark of natural aphrodisiacs such as
P. yohimbe and R. serpentina with recoveries in the range of 9297%.
In order to facilitate a high throughput of samples to be analyzed,
ow-injection analysis (FIA) technique coupled with amperometric detection presents favorable characteristics, such as simplicity,
good sensitivity, low cost and consumption of reagents, and relatively short analysis time [19]. This system has been widely used
for the sensitive determination of various species [20,21]. The current report shortly describes the development of a novel, simple
and rapid FIA methodology for the sensitive amperometric determination of YOH on BDD electrode. In addition, there have been no
papers for the FIA determination of YOH and related alkaloids on
BDD electrodes published in the literature until now.
2. Experimental
Yohimbine hydrochloride (purity 98%, YOH) was purchased
from SigmaAldrich (Austria) and used without any further purication. Its stock standard solution was prepared by dissolving
it in deionized water; it was stored in a refrigerator at 46 C.
BrittonRobinson buffer solution was prepared by mixing of phosphoric acid, acetic acid and boric acid (all at 40 mM) and adjusting
the pH with sodium hydroxide (0.2 M) to the desired value. All other
chemicals were of analytical reagent grade. Deionized water with
resistivity not less than 18 M cm (Millipore Milli-Q system) was
used for preparation of all solutions.
The ow-injection system consisted of a peristaltic pump (510
Waters, Milford, MA, USA), a sample injection valve (MX7925 Rheodyne, Cotati, USA), and a home-made amperometric thin-layer ow

Fig. 1. (A) Hydrodynamic voltammogram of 0.1 mM YOH obtained by a FIA system

with a BDD electrode as detector in BRBS at pH 7; injection volume 100 L, ow
rate 1.4 mL min1 ; YOH 0.1 mM. (B) The dependence of the signal on pH; all data are
mean values of three measurements.

cell in the three-electrode conguration in combination with an

electrochemical workstation (BAS 100B). The thin-layer amperometric ow cell was constructed with the BDD electrode (Windsor
Scientic Ltd., UK; inner diameter 3 mm, resistivity of 0.075  cm,
boron doping level 1000 ppm), which was inserted into a whole of
an in house-made Teon plate acting as the back part of the cell,
a spacer (0.15 mm), a miniaturized Ag/AgCl (3 M KCl) as reference
electrode, and a steel back plate served as the counter electrode.
Prior to use at the beginning of every work way, the bare BDD
electrode was rinsed with deionized water and rubbed very gently
with a piece of damp silk cloth until a mirror-like appearance of
surface was obtained. Subsequently before inserting into amperometric ow cell, it was anodically pretreated by applying +2 V for
60 s in 0.5 M H2 SO4 solution in order to clean the electrode surface (get rid of any impurities) followed by cathodic pretreatment
at 2 V for 60 s to retain a predominantly hydrogen-terminated
surface. Such electrochemically pretreated working electrode provided the reliable current response during the whole work day.
Hydrodynamic voltammograms were recorded at potentials from
+0.6 to +1.5 V with an increment of 0.1 V. Flow-injection analyses
were performed at a potential of +1.2 V with constant injection
volume of 100 L if not stated otherwise. The program OriginPro
8.0 (OriginLab Corporation, USA) was used for the evaluation of
the data. The detection and quantication limit were calculated as
three and ten times the standard deviation for the blank solution
divided by the slope of the calibration curve.
Dietary supplements (primary bark from P. yohimbe and R. serpentina) were purchased in a local shop in Vienna (Austria). One
gram of powdered bark was macerated with 20 mL methanol and
evaporated to dryness at 70 C. The residue was dissolved in 30 mL
HCl (2%, w/w) and ltered. The pH of the ltrate was adjusted to
7 with 0.1 M NaOH; it was then extracted three times with 20 mL
chloroform. The chloroform layers were combined and evaporated
to dryness; the residues were subsequently dissolved in 20 mL
methanol and ltered [10]. The extract was made up to 50 mL with
supporting electrolyte in a volumetric ask. A 100 L aliquot of
the sample was injected into the FIA system; concentrations were
evaluated with the calibration curve.
3. Results and discussion
YOH is a biologically, structurally and electrochemically interesting indole alkaloid capable of transferring electrons from the
solution to the electrode surface. Fig. 1 displays the hydrodynamic voltammogram (HDV) for the electrochemical oxidation of

L. S vorc, K. Kalcher / Sensors and Actuators B 205 (2014) 215218

217

Table 1
Analytical characteristics for FIA amperometric determination of YOH using proposed method (n = 3).
Analytical parameters
Detection potential (V vs. Ag/AgCl)
Sampling frequency (injections h1 )
Linear concentration range (M)
Intercept (nA)
Standard deviation of intercept (nA)
Slope (nA M1 )
Standard deviation of slope (nA M1 )
Coefcient of determination (R2 )
Precisiona (RSD %)
Detection limit (M)
Quantication limit (M)
a

Fig. 2. Flow-injection amperograms of various concentrations of YOH at optimized

experimental conditions: detection potential of +1.20 V, ow rate of 1.4 mL min1 ,
injection volume of 100 L and BRBS at pH 7. The calibration curve appears in the
inset (error bars from n = 3).

0.1 mM YOH in BrittonRobinson buffer solution (BRBS) at pH

7 in a FIA system. The HDV was obtained by plotting the peak
current (mean value of three injections) as a function of the corresponding applied detection potential (Edet ) using BDD as a working
electrode with an injection volume of 100 L and a ow rate of
1.4 mL min1 .
As can be seen, the oxidation current of YOH started to increase
at approximately +0.7 V and leveled off at +1.2 V with maximum
value at +1.5 V (330 nA). However, detection potentials from +1.3 to
+1.5 V yielded worse-shaped responses with high backgrounds and
large current uctuations. Hence, in order to minimize the potential
interferences of other species (alkaloids) oxidizing at higher positive potentials and to maintain a relative low background signal,
a detection potential of +1.2 V was selected as the most suitable
in all subsequent FIA measurements. The electrochemical behavior was also investigated in phosphate and acetate buffers showing
similar characteristics of the HDVs, however, with lower oxidation
currents (results not shown). Therefore, based on this fact, BRBS
buffer was adopted as an appropriate supporting electrolyte for
the determination of YOH using FIA system.
The effect of pH on the oxidation current of 0.1 mM YOH was
studied by FIA measurements in the pH range of 212 (BRBS) with
a detection potential of +1.2 V and ow rate of 1.4 mL min1 . The
obtained results indicated a slight enhancement of the current of
YOH when increasing the pH from 2 to 5; beyond pH 5 it started to
increase sharply and reached a maximum at pH 7 beyond which it
decreased sharply as depicted in the inset of Fig. 1. Therefore, pH
7 represented the most suitable pH of the supporting electrolyte
for the FIA amperometric determination of YOH. The inuence of
the ow rate in the range from 0.6 to 1.4 mL min1 was also examined. Increasing the ow caused a diminution of the diffusion layer
thickness on the BDD electrode surface consequently providing an
increase of the diffusion current with narrower peaks. Additionally, also the dispersion of the analyte, due to its residence in the
streaming bulk, is less pronounced with higher velocities of the
carrier. A maximum value was obtained above the ow rate of
1.4 mL min1 that was selected for further. Overall, BRBS at pH 7,
a detection potential of +1.2 V, a ow rate of 1.4 mL min1 with a
constant injection volume of 100 L were considered to be optimum experimental conditions for the quantication of YOH in FIA
measurements.
Once the most suitable experimental conditions for the determination of YOH were established, FIA measurements at different
concentrations of YOH were carried out to examine the analytical
performance of proposed method. Fig. 2 depicts FIA amperograms

Value
+1.20
70
0.310
3.0
0.3
6.2
0.2
0.995
4.5
0.15
0.50

10100
40
4
2.1
0.1
0.992

Calculated for 10 replicate FIA measurements at 10 M YOH.

recorded for injections of 100 L of standard solutions (in triplicate) containing increasing concentrations of YOH (from 0.3 to
100 M). The respective calibration curve with the obtained linear
relationship between oxidation current and concentration of YOH
is presented in the inset of Fig. 2.
It is evident that the calibration graph consists of two linear
segments, from 0.3 to 10 M and from 10 to 100 M with good
linearity. The mean data of both segments of calibration curve,
statistical evaluation of the regression lines and the analytical characteristics for the developed method are summarized in Table 1.
Above 100 M the signal levels off.
The detection limit was calculated to be 0.15 M (0.053 mg L1 )
as a consequence of the high S/N ratio using rst linear segment
(0.3-10 M) of the calibration graph. The precision (repeatability)
of the proposed method was evaluated by ten replicate injections
of 10 M YOH under the same operating conditions over the short
time interval. The relative standard deviation (RSD) was calculated as 4.5% conrming a good repeatability of the procedure and
demonstrating only minimal adsorption of components onto the
detector surface present in the analyzed solution. The sampling
frequency was calculated as about 70 injections h1 .
To investigate the possibility of applying proposed FIA methodology for the amperometric determination of YOH, an interference
study was performed for assessing the matrix effect on the current
response of YOH. Structurally related indole alkaloids such as reserpine and ajmalicine were being considered to be most signicant
interfering agents in the bark samples. However, a more detailed
amperometric study of these alkaloids under FIA conditions with
the BDD electrode using BRBS at pH 7 revealed their electrochemical inactivity. The results of interference study showed that a
20-fold excess of reserpine and ajmalicine had negligible effect on
the oxidation current of YOH. Thus, the proposed procedure provides good selectivity for the amperometric determination of YOH.
The extracts from dietary supplements (primary bark from P.
yohimbe and R. serpentina) were analyzed in order to evaluate the
validity and the practical applicability of the developed FIA procedure. The recovery analysis was performed in order to estimate
the accuracy of the proposed method and to know whether the
ingredients present in the analyzed extracts show any interferences. The results are summarized in Table 2. The recovery values
ranged from 94 to 98% and from 92 to 95% for P. yohimbe and R. serpentina, respectively demonstrating that there are no signicant
matrix interferences in the analyzed samples. Thus, YOH can be
quantitatively determined by the proposed method, being thus a
guarantee of the accuracy and suitability of the determination of
YOH in the samples of this kind.
In conclusion, we successfully developed a sensitive FIA procedure with amperometric detection using a BDD electrode as
working electrode for the determination of YOH in dietary supplements. The proposed method is considerably more cost-effective

L. S vorc, K. Kalcher / Sensors and Actuators B 205 (2014) 215218

218

Table 2
Recovery analysis of YOH in the extracts of Pausinystalia yohimbe and Rauvola serpentina using proposed method (n = 3).
Sample

Found (M)

Added (M)

Determineda (M)

11.5

10
20
30
10
20
30

20.2
30.9
40.7
21.9
30.8
41.3

Pausinystalia yohimbe
13.5
Rauvola serpentina
a

0.6
0.5
0.7
0.3
0.3
0.5

Recovery (%)
94
98
98
93
92
95

Condence interval: [mean tn1 , SD/sqrt(n)]; t2,0.05 = 2.92.

than separation methods, especially HPLC and GC techniques. The

analytical performance of herein described method is comparable
with that recently achieved on BDD electrode in batch arrangement
with the linear concentration range from 0.25 to 90.9 M and the
detection limit of 0.13 M [18]. However, the signicance of proposed FIA methodology embodies in rapid determination of YOH
with sampling frequency about 70 injections h1 . The developed
method renders the assessment of YOH traces with the detection
limit of 0.15 M which is 6 and 300-fold higher when compared
to the HPLC method with amperometric [14] and coulometric
detection [15], respectively. However, it could be taken into consideration that these combined techniques have been explicitly
developed for pharmacokinetic study of YOH and other indole alkaloids in biological samples. In addition, our approach primarily lies
in the quantication of YOH in P. yohimbe and R. serpentine. In
this sense, the BDD electrode fullls the requirements for possible applications in FIA systems as sensitive amperometric sensor
and can be easily adapted for quality control analysis of dietary
supplements containing various alkaloids.
Acknowledgements
This work has been supported by the Grant Agency of the Slovak
Republic (grant no. 1/0051/13), the Slovak Research and Development Agency under the contract no. APVV-0797-11. Support of the
bilateral program Action: Austria-Slovakia is gratefully acknowledged.
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Biographies
Dr. Lubomr S vorc is a young researcher under the age of 35. He was graduated
in 2006 and received his PhD in 2009 in Analytical Chemistry in Slovak University
of Technology in Bratislava, Slovak Republic. His interest covers the development
and characterization of new electrode materials for application of electrochemical
sensors and biosensors based on boron-doped diamond for solving the tasks of food,
clinical and environmental trace analysis. At his young age, he is author and coauthor of more than 60 publications.
Ao.Univ.-Prof. Dr. Kurt Kalcher is the head of the work group Electroanalysis and
Sensorics of the Institute of Chemistry-Analytical Chemistry of the Karl-Franzens
University in Graz, Austria. His main scientic interest lies in the development of
electrochemical sensors and biosensors based on carbon paste and other carbonaceous materials. Besides one of another focus is the development of simple analytical
devices including microprocessor control and data acquisition and handling. He has
authoredcoauthored around 200 publications.