92 Acta Chim. Slov. 2007, 54, 92–97
Mikelová et al.:   Determination of Isoflavones Using Liquid Chromatography with Electrochemical Detection
1. Introduction
Nutraceutics are food components having a positive
physiological impact on human organism.1 Certain nutra-
ceutics are beneficial to human health, others increase
physical performance or decrease the risk of illnesses.
From the chemical point of view, nutraceutics include a
huge number of chemically different compounds, and inc-
lude flavonoids. The interest in biological activity of fla-
vonoids, estrogen-like compounds of plant origin, started
only recently.2,3 Flavonoids comprise a wide-ranging
group of plant phenols. Up to now, more than 4,000 flavo-
noid compounds have been described.4–7 They are derived
from the oxygen-containing heterocyclic compound fla-
van. These phytoestrogens encompass several classes of
compounds, including flavonoids, isoflavonoids, coume-
strans (coumestrol), lignans and isoflavones. Isoflavones
represent a group of distinct secondary metabolites produ-
ced predominantly in leguminous plants. Different isofla-
vones are derived through oxidation of the three central
atoms in the carbon skeleton of 3-phenylbenzpyrone.
Their structures differ in the degree of methylation,
hydroxylation and glycosylation (Figure 1).
Isoflavones can be considered to be biologically im-
portant compounds. Isoflavones (e.g. daidzein) are pre-
cursors of phytoalexins and have a role in plant disease re-
sistance, and exhibit antifungal activity (e.g. genistein).2
The role of these compounds in carcinogenetic processes
has been investigated intensively, although it has not been
clarified yet.8–11 It has also been published that the biolo-
gically active derivatives of isoflavones could be formed
by action of cytochromes.12–14
A wide range of analytical techniques have been
used for determination of polyphenols and phytoestrogens
in food and biological materials.15–20 Electrochemical
techniques and methods are an attractive alternative met-
Abstract
Among the biologically important roles of isoflavones is also their effect on carcinogenesis. We used flow injection
analysis and high performance liquid W with electrochemical detection to simultaneously determine certain isoflavones
(biochanin A, formononetin, sissotrin, daidzin, daidzein, glycitin, glycitein and genistein). The most suitable chromato-
graphic conditions were: mobile phase: 0.2 mol L–1 acetate buffer (pH 5.0); flow rate 2.0 mL min–1; column and detec-
tor temperature: 26 °C; detection potential: 800 mV. Under the optimal conditions, the detection limits were in the ran-
ge of several ng mL–1. Their simultaneous determination takes 15 min.
Keywords: isoflavone, electrochemical detection, coulometry, estrogen-like compounds, carbon paste electrode
Short communication
Determination of Isoflavones Using Liquid 
Chromatography with Electrochemical Detection
Radka Mikelová,a Petr Hodek,b Pavel Hanustiak,c,d Vojtech Adam,b
Sona Krizkova,b Ladislav Havel,e Marie Stiborova,b Ales Horna,f
Miroslava Beklova,d Libuse Trnkova,a Rene Kizekc,*
a Masaryk University, Department of Theoretical and Physical Chemistry, Faculty of Science, Brno, Czech Republic
b Charles University, Department of Biochemistry, Faculty of Science, Prague, Czech Republic
c Mendel University of Agriculture and Forestry, Department of Chemistry and Biochemistry, and 
e Department of Plant Biology, Faculty of Agronomy, Czech Republic. Tel.: +420(5)45133350, 
Fax: +420(5)45212044, E-mail: kizek@sci.muni.cz.
d University of Veterinary and Pharmaceutical Sciences, Department of Veterinary Ecology 
and Environmental Protection, Brno, Czech Republic
f Tomas Bata University, Department of Food Engineering and Chemistry, Faculty of Technology, Zlin, Czech Republic
Received: 09-10-2006
Paper based on a presentation at the 12th International Symposium on Separation Sciences, 
Lipica, Slovenia, September 27–29, 2006.
93Acta Chim. Slov. 2007, 54, 92–97
Mikelová et al.:   Determination of Isoflavones Using Liquid Chromatography with Electrochemical Detection
hod for detection of electroactive species, because of sim-
plicity, ease of miniaturization, high sensitivity and relati-
vely low cost.21–29
Here, we optimized and utilized flow injection
analysis and high performance liquid chromatography
coupled with electrochemical detection to determine bioc-
hanin A, formononetin, sissotrin, daidzin, daidzein, glyci-
tin, glycitein, genistein, simultaneously.
2. Experimental
2.1. Chemicals
Isoflavones (biochanin A, formononetin, sissotrin,
daidzin, daidzein, glycitin, glycitein, genistein) were
purchased from Karlsroth GmbH (Karlsruhe, Germany).
HPLC-grade acetonitrile (>99.9%) was from Merck
(Darmstadt, Germany). Acetate buffer, trifluoroacetic acid
and other analytical reagents of ACS purity were purcha-
sed from Sigma Aldrich (St. Louis, USA) unless noted ot-
herwise. The stock standard solutions of isoflavones (10
mg mL–1) were prepared in methanol (Sigma Aldrich,
USA) : water 1:1 v/v and stored in darkness at 4 °C. The
working standard solutions were prepared daily by dilu-
tion of the stock solutions. All solutions were filtered
through a 0.45 µm teflon membrane filters (MetaChem,
Torrance, CA, USA) prior to HPLC analysis.
2.2. Instrumentation
The flow injection analysis/high performance liquid
chromatography coupled with electrochemical detector
(FIA-ED/HPLC-ED) system consisted of two solvent de-
livery pumps operating in range of 0.001–9.999 mL min–1
(Model 582 ESA Inc., Chelmsford, MA), a reaction loop
1 m long and an electrochemical detector (Model 5600A,
ESA, USA). Zorbax AAA reversed-phase chromatograp-
hic column (150 mm × 4.6 mm, 3.5 µm particle size, Agi-
lent, USA) was used instead of reaction loop to determine
the isoflavones simultaneously. The electrochemical de-
tector includes two low volume flow-through analytical
cells (Model 6210, ESA, USA). Each analytical cell is
consisted of four carbon porous working electrodes, palla-
dium electrodes as reference ones and auxiliary electro-
des. The detector and the column were thermostated. The
sample (5 µL) was injected manually (Hamilton, USA).
Figure 1. Chemical structures of biochanin A, formononetin, sissotrin, daidzin, daidzein, glycitin, glycitein, and genistein.
94 Acta Chim. Slov. 2007, 54, 92–97
Mikelová et al.:   Determination of Isoflavones Using Liquid Chromatography with Electrochemical Detection
2.3. Statistical Analysis
STATGRAPHICS® (Statistical Graphics Corp®,
USA) was used for statistical analyses. Results are expres-
sed as the means ±S.D. unless noted otherwise. Value of p
< 0.05 was considered significant.
3. Results and Discussion
3.1. Flow Injection Analysis
As we already demonstrated, using flow injection
analysis coupled to various detectors we can investigate
the behaviour of compounds of interest in order to optimi-
se detection.30–33 Here, we focused on the optimisation of
detection of isoflavones in the range of applied potential
300 to 1000 mV. The experiments were carried using ace-
tate buffer pH 5.0 as a mobile phase with flow rate of 0.5
mL min–1; temperature 20 °C. The hydrodynamic voltam-
mograms are shown in Figure 2. The isoflavones of inte-
rest gave the highest responses at potentials within the
range of 750 to 900 mV. The signals at the same concen-
tration (100 µmol L–1) were similar, except daidzein, whe-
re the response was about ten times higher. Based on the-
se results, the potential 800 mV was chosen for the opti-
misation to follow.
3.2. Influence of the Mobile Phase 
Composition
The mobile phase composition markedly affects
electrochemical determination of the compounds of inte-
rest.34 Two commonly used mobile phases, 0.2 mol L–1
acetate buffer and 0.05 mol L–1 trifluoroacetic acid (TFA),
were tested. We observed an interesting electrochemical
response. In the case of acetate buffer, the observed an in-
crease of signal with an increasing pH. If we used TFA,
we observed a decrease of the signal with an increasing 
pH (Figure 3). This behaviour was common to all iso-
flavones. The highest signals were obtained using 
0.2 mol L–1 acetate buffer at pH 5 (Figure 3C).
Figure 2. FIA-ED hydrodynamic voltammograms of the isoflavones of interest: peak heights (A) and cumulative responses (B). Inset: hydrodyna-
mic responses for daidzein. Mobile phase: 0.2 mol L–1 acetate buffer (pH 5.0); concentration of isoflavones 100 µmol L–1.
95Acta Chim. Slov. 2007, 54, 92–97
Mikelová et al.:   Determination of Isoflavones Using Liquid Chromatography with Electrochemical Detection
3.3. Influence of the Flow Rate 
and Temperature
Flow rate within the range of 1–3 mL min–1 was
investigated. At higher flow rates, signals of the isofla-
vones increased (Figure 4A). The optimal flow of mobi-
le phase was 2 mL min–1 at 20 °C. The influence of de-
tector temperature is shown in Figure 4B. The current
response of isoflavones increased with temperature up
to 30 °C, then we observed a slight decrease of the sig-
nal of 10–15%. On the other hand, a higher standard de-
viation (>5%) was observed at 30 °C. Therefore, we
chose the detector temperature of 26 °C as the most sui-
table, where the relative standard deviation was ∼3.5%
(n = 5).
3.4. Calibration Curves
At the optimized conditions, the relationship bet-
ween signal intensity and isoflavone concentration was
determined. The calibration curves were linear within the
concentration range of 50–1000 ng mL–1 (R2 = 0.99), see
Table 1. The relative standard deviation varied from
2.4–4.1%.
Figure 3. FIA-ED dependence of peak heights on pH of the mobile phase: 0.2 mol L–1 acetate buffer (A), 0.05 mol L–1 trifluoroacetic acid (B), inf-
luences on cumulative peak heights (sum of peak heights of the individual isoflavone at the respective pH) of the studied isoflavones (C). Potentials
of all electrodes: 800 mV (peak height and area was determined from peaks at the first detector electrode). Other details see Figure 3.
Figure 4. Dependence of peak heights and cumulative peak heights of isoflavones of interest at different flow rates (A) and temperature (B). For ot-
her details see Figure 3.
3.5. HPLC-ED
To ensure the separation of isoflavones in 30 min, an
addition of organic solvent in the mobile phase was nee-
ded, not exceeding 35% (v/v) due to a decrease in sensiti-
vity. The separation and sensitivity of the method are sa-
tisfactory (Figure 5).
4. Conclusion
We present a simple, easy-to-use and low-cost
technique enabling us to determine eight various isofla-
vones (biochanin A, formononetin, sissotrin, daidzin,
daidzein, glycitin, glycitein and genistein) within 15 min
and with satisfactory detection limits. This technique
could be used for routine analysis, as almost no sample
preparation is needed and due to the low associated
costs.
5. Acknowledgement
This work was supported by grants GACR
525/04/P132, MSMT 6215712402, INCHEMBIOL
0021622412 and MSMT 1M06030.
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96 Acta Chim. Slov. 2007, 54, 92–97
Mikelová et al.:   Determination of Isoflavones Using Liquid Chromatography with Electrochemical Detection
Figure 5. HPLC-ED chromatogram of
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Table 1. Method parameters for determination of isoflavones (n = 5). Applied potential: 800 mV.
Isoflavones Regression equation R2,a LODb LODb LODc LOQd LOQd R.S.D.e
(ng mL–1) (nmol L–1) (fmol) (ng mL–1) (nmol L–1) (%)
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Genistein y = 15.90x + 0.63 0.9917 0.41 1.5 7.7 1.4 5.1 3.2
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c … Limits of detection per
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d … Limits of quantification 
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Povzetek
Med biolo{ko pomembnimi funkcijami izoflavonov je tudi njihov u~inek na karcinogenezo. Za simultano analizo neka-
terih izoflavonov (biohanin A, formononetin, sisotrin, daidzin, daidzein, glicitin, glicitein in genistein) smo uporabili
preto~no analizo in teko~insko kromatografijo v povezavi z elektrokemijsko detekcijo. Kot najprimernej{o mobilno fa-
zo predlagamo 0,2 mol L–1 acetatni pufer (pH 5,0) pri pretoku 2,0 mL min–1, temperaturi kolone in detektorja 26 °C in
potencialu na detektorju 800 mV. Pri optimalnih pogojih je meja dolo~ljivosti nekaj ng mL–1.