Highly Sensitive Determination of Hesperidin Using Electrode Modified with Poly(Ferulic Acid) ^†

Abstract

Hesperidin is a major phenolic antioxidant in orange fruits and is responsible for their positive health effect. It is used as part of the therapy for blood vessel conditions. Methods for hesperidin quantification are of practical interest. Recently, several voltammetric approaches have been developed for hesperidin quantification. Nevertheless, the analytical characteristics could be improved. To solve this problem, a glassy carbon electrode (GCE) modified with multi-walled carbon nanotubes (MWCNTs) and poly(ferulic acid) has been developed. Polymeric coverage has been obtained electrochemically under potentiodynamic conditions. Their optimization based on the hesperidin voltammetric response has been performed. The poly(ferulic acid) layer has to be obtained from 250 µM monomer solution in 0.1 M NaOH by fifteen potential scans from −0.2 to 1.0 V with the scan rate of 100 mV s⁻¹. Hesperidin oxidation currents are 2.8-fold increased at the polymer-modified electrode vs. carbon nanotube-based electrode at the same oxidation potential. Differential pulse voltammetry in phosphate buffer pH 5.5 has been used for the quantification of hesperidin. Linear dynamic ranges of 0.025–1.0 µM and 1.0–10 µM have been achieved with the limits of detection and quantification of 7.0 and 23.4 nM, respectively. The analytical characteristics obtained are the best ones reported to date.

1. Introduction

Hesperidin (hesperetin-7-O-rutinoside) is a major phenolic antioxidant in orange fruits and is responsible for their positive health effect [1]. It is used as part of the therapy for blood vessel conditions [2,3]. Nevertheless, overdose of hesperidin can lead to harmful effects. On the other hand, the hesperidin content in orange fruits and juices is one of the parameters characterizing their nutritional value. Therefore, control of hesperidin in oranges is of interest and simple methods for hesperidin quantification are required.

Hesperidin is an electroactive compound that necessitated the development of voltammetric approaches for hesperidin quantification. Recently, boron-doped diamond [4] and disposable pencil graphite (bare [5] and electroactivated [6]) electrodes have been developed for the determination of hesperidin. To improve the sensitivity and selectivity of hesperidin response, several chemically modified electrodes have been fabricated using carbon nanomaterials [7,8,9], SnO₂ [10], mesoporous SiO₂ [11], Au [12] nanoparticles, polymeric films based on polyaluminon [13] and molecularly imprinted poly-o-aminothiophenol [14] as a sensitive layer. The analytical characteristics of hesperidin at these electrodes are not impressive (linear dynamic ranges of n × 10⁻⁸–n × 10⁻⁵ M or even worse with the limits of detection n × 10⁻⁸–n × 10⁻⁶ M) and can be further improved.

The current work deals with the development of a novel modified electrode based on the combination of MWCNTs and electropolymerized ferulic acid. Such a type of modification has shown effectivity in the electroanalysis of natural phenolic antioxidants [15] and structurally related naringin [16]. This type of modifier is novel for hesperidin. The conditions of ferulic acid electropolymerization at the GCE covered with MWCNTs have been optimized. A highly sensitive voltammetric approach for hesperidin quantification has been developed.

2. Materials and Methods

The 0.40 mM stock solution of hesperidin was prepared in methanol (c.p. grade) using 94% hesperidin from Sigma (Steinheim, Germany). Less concentrated solutions were obtained by exact dilution.

MWCNTs (outer diameter 40–60 nm, inner diameter 5–10 nm and 0.5–500 μm length) from Aldrich (Steinheim, Germany) were used as a platform for further electrodeposition of poly(ferulic acid). Homogeneous 0.5 mg mL⁻¹ suspension of MWCNTs was prepared in 1% sodium dodecylsulfate (Panreac, Barcelona, Spain) by 30 min of sonication in an ultrasonic bath WiseClean WUC-A03H (DAIHAN Scientific Co., Ltd., Wonju-si, Republic of Korea). The GCE surface was polished on 0.05 µm alumina slurry and rinsed with acetone and distilled water. Then, 5 µL of MWCNTs suspension was drop casted on the electrode surface.

All reagents were c.p. grade. Distilled water was used for the measurements. The laboratory temperature was (25 ± 2 °C).

Electrochemical measurements were conducted on the potentiostat/galvanostat Autolab PGSTAT 12 (Eco Chemie B.V., Utrecht, The Netherlands) with the NOVA 1.10.1.9 software (Eco Chemie B.V., Utrecht, The Netherlands). The glassy electrochemical cell of 10 mL volume was used. The three-electrode system consisted of the working GCE of 3 mm diameter (BASi^® Inc., West Lafayette, IN, USA), or a modified electrode, an Ag/AgCl reference electrode and a platinum wire as the auxiliary electrode.

The pH measurements were carried out using the “Expert-001” pH meter (Econix-Expert Ltd., Moscow, Russia) with a glassy electrode.

3. Results and Discussion

Electrodeposition of poly(ferulic acid) was performed in a potentiodynamic mode in a basic medium, providing easier oxidation of the monomer. There is a clear irreversible oxidation step at 0.31 V of the cyclic voltammograms of ferulic acid (Figure 1) corresponding to the one electron detachment from the phenolate ion with the formation of a phenoxyl radical (Scheme 1) that undergoes further reactions of dimerization and polymerization, similar to other hydroxycinnamic acids [15]. Oxidation currents are significantly decreased on the following scans, which is caused by the formation of insulating coverage and agrees well with reported values of other phenolic acids [15].

Since the electropolymerization conditions affect the properties of the polymeric coverage obtained, the optimization of poly(ferulic acid) deposition has been performed on the basis of the hesperidin response. The poly(ferulic acid) layer has to be obtained from the 250 µM monomer solution in 0.1 M NaOH by fifteen potential scans from −0.2 to 1.0 V with a scan rate of 100 mV s⁻¹.

The polymer-modified electrode provides a statistically significant increase in hesperidin oxidation currents in comparison with an electrode based on MWCNTs and an unmodified electrode (

Table 1. Voltammetric characteristics of 10 μM hesperidin at various electrodes in phosphate buffer pH 7.0 on the basis of differential pulse voltammetry data.

Electrode	Eox1 (V)	Iox1 (μA)	Eox2 (V)	Iox2 (μA)
GCE	0.563	0.056 ± 0.002	0.935	0.045 ± 0.002
MWCNTs/GCE	0.504	0.18 ± 0.01	0.886	0.23 ± 0.01
Poly (ferulic acid)/MWCNTs/GCE	0.509	0.50 ± 0.02	0.904	0.42 ± 0.02

). These data confirm the effectivity of the modified electrode developed in hesperidin sensing.

Differential pulse voltammetry has been applied for hesperidin quantification. The variation of phosphate buffer pH has shown that the best hesperidin response occurs at pH 5.5. A further increase in pH leads to a decrease in the oxidation currents, which is caused by the partial oxidation of hesperidin by air oxygen that is typical for flavonoids [17].

The effect of the pulse parameters on the hesperidin response has been evaluated. The oxidation potential is insignificantly decreased as pulse amplitude and time are increased. The oxidation currents are changed statistically significantly. The increase of pulse amplitude provides growth of the hesperidin oxidation currents, which achieves maximum at a pulse amplitude of 100 mV. An increase in the pulse time from 25 to 100 ms leads to a significant decrease in the oxidation currents. Thus, pulse amplitude of 100 mV and pulse time of 25 ms have been chosen for further measurements.

There are two well-resolved oxidation peaks of hesperidin on the voltammograms (Figure 2).

The first oxidation step has been used as an analytical signal. Linear dynamic ranges of 0.025–1.0 µM and 1.0–10 µM have been achieved with limits of detection and quantification of 7.0 and 23.4 nM, respectively. The analytical characteristics achieved are the best among those reported to date.

The accuracy of the method developed has been tested by the added–found method using model solutions of hesperidin (

Table 2. Determination of hesperidin in model solutions at the poly(ferulic acid)/MWCNTs/GCE in phosphate buffer pH 5.5 (n = 5; p = 0.95).

Added Amount (µg)	Found Amount (µg)	RSD (%)	R (%)
0.0610	0.061 ± 0.002	3.2	100
0.610	0.61 ± 0.01	1.6	100
2.44	2.43 ± 0.04	1.4	99.6
12.2	12.2 ± 0.1	0.79	100
24.4	24.3 ± 0.3	1.0	99.6

). The recovery of 99.6–100% indicated a high accuracy of hesperidin determination. The relative standard deviation of 0.79–3.2% confirms the absence of random errors in the determination and the good reproducibility of the electrode response, since the electrode surface has been renewed after each measurement.

4. Conclusions

A sensitive voltammetric method for the determination of hesperidin has been developed using an electrode modified with MWCNTs and poly(ferulic acid). The obtained analytical characteristics are significantly improved compared to those described earlier for other electrodes including chemically modified ones. The method is characterized by rapidity, simplicity and reliability of the results obtained.