Voltammetric Determination of Vitamin B 6 at Glassy Carbon Electrode Modified with Gold Nanoparticles and Multi-Walled Carbon Nanotubes

In this work, the gold nanoparticles (Au NPs)/multi-walled carbon nanotubes (MWCNTs) composite film modified glassy carbon electrode (GCE) was fabricated, and scanning electron microscopy (SEM) was used to investigate the assemble process of the composite film. In pH 7.0 PBS, an oxidation peak of the vitamin B6 (VB6) was only observed at composite film modified electrode. Under the optimized conditions, the current intensity was linear with the concentrations of VB6 in the range of 1.59 to 102.74 μg·mL with a detection limit of 0.53 μg·mL (S/N = 3). The modified electrode had been applied in pharmaceutical analysis, and obtained good results.


Introduction
Vitamin B 6 (VB 6 ), also known as pyridoxine, is part of the B group vitamins and plays an important role in the synthesis and metabolism of amino acid.The deficiency of VB 6 has been suggested as the cause of many types of illness and disease [1].Several analytical methods have been described in literature for determination of VB 6 , including spectrophotometry [2,3], liquid chromatogramphy [4,5] and electrochemistry method [6,7].R. Jiménez-Prieto et al determined VB 6 by spectrophotometric techniques in the presence of other vitamins [8].Fang et al [9] detected VB 6 in pharmaceutical preparations by micellar electrokinetic chromatography with amperometric electrochemical method.Söderhjelm and Lindquist [10] firstly detected VB 6 in pharmaceutical preparations with electrochemical method; however, the interferences from ascorbic acid were existed and had not been repelled.Until now, few papers had been described on the determination of VB 6 using electrochemical method.
Carbon nanotubes (CNTs) have been an important group of nanomaterials with attractive geometrical, electronic and chemical properties.Recently, composite materials based on integration of CNTs and other materials had gained growing interest, including conducting polymers, redox mediators and metal nanoparticles [11][12][13][14].Gold nanoparticles (Au NPs) were another important nanomaterial, and it had also been used in biosensor of glucose [15,16], DNA [17], pesticides [18], and arsenic (III) [19].We have used Au NPs/CNTs composite film modified electrode to detect Sal and target DNA [20,21] and obtained good results.From our practical experiment, we find the Au NPs/CNTs composite are good materials, and hope to open more applied window.We want to study medicament analysis based on this ideas.To our knowledge, there was no report of VB 6 detection based on Au NPs and MWCNTs composite film modified electrode.
In this work, Au NPs was electrodeposited on the surface of the MWCNTs modified electrode, and the electrochemical behavior of VB 6 at this modified electrode was investigated, the experiment results showed an oxidation peak of VB 6 was only observed in CVs and the electrochemical response of VB 6 was higher in contract to MWCNTs or Au NPs modified electrode alone.Under optimal experiment condition, the current intensity was linear with the concentrations of VB 6 in the range of 1.59 to 102.74 μg•mL -1 with a detection limit of 0.53 μg•mL -1 (S/N = 3).When the modified electrode was used to detect VB 6 in pharmaceutical preparations, a satisfied result was obtained.

Reagents
Multi-walled carbon nanotubes with carboxylic acid groups (MWCNTs, with a diameter of about 30 nm and length of around 30 μm, purity > 95%) were obtained from Chengdu Institute of Organic chemistry, Chinese Academy of Sciences.Vitamin B 6 , Vitamin B 1 (VB 1 ), Vitamin B 2 (VB 2 ), Ascorbic acid (AA), and HAuCl 4 •4H 2 O were obtained from Shanghai Chemical Reagent Co., Ltd (China).The phosphate buffer solution (PBS, 0.10 mol•L -1 ) was prepared by NaH 2 PO 4 and Na 2 HPO 4 , and adjusted the pH with H 3 PO 4 and NaOH solutions.All chemicals were of analytical grade and used without further purification.All solutions were prepared with twicequartz-distilled water.

Apparatus
Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed on a CHI 650C electrochemical workstation (Shanghai Chenhua Instruments Co., China).The three-electrode system consisted of a bare GCE or modified electrode as a working electrode, a platinum wire as a counter electrode, and a saturated calomel electrode (SCE) as a reference electrode.All potentials given in this work were referenced to the SCE reference electrode.
The morphologies of different modified electrodes were obtained using a scanning electron microscopy (SEM) with JEOL JSM-4800F microscopy (Hitachi, Japan).

Preparation of the Au NPs/MWCNTs/GCE
The bare GCE was carefully polished sequentially with 0.3 and 0.05 m alumina slurries on microcloth pads followed by rinsing successively in an ultrasonic bath with acetone, absolute alcohol and twice-quartz-distilled water for 3 min, respectively.Afterwards, the electrode was electrochemically treated by cycling the potential between − 0.3 and + 1.5 V in 0.50 mol•L -1 H 2 SO 4 with a scan rate of 100 mV•s -1 until a reproducible cyclic voltammogram was observed, followed it is rinsed with twice-quartz-distilled water thoroughly and dried in a nitrogen stream.
5 µL of 1.0 mg•mL -1 MWCNTs suspensions (10.0 mg of MWCNTs was dispersed in 10.0 mL of ethanol with the help of ultrasonication) were dropped on the surface of pretreated GCE, and then it was dried at room temperature.The Au NPs/MWCNTs/GCE was obtained by immersing the MWCNTs/GCE into 0.10 mol•L -1 KNO 3 solution containing 1 mmol•L -1 HAuCl 4 and electrodeposited 20 s at -0.2 V (vs.SCE).

Procedure
The preparation of sample: 1.0 mL of injection (Henan Topfond Pharmaceutical Co., Ltd.) was transferred to a 10.0 mL volumetric flask, diluted to the mark with twicequartz-distilled water.Next, 2.0 mL of solution above was taken to 10.0 mL volumetric flask, diluted to the mark with twice-quartz-distilled water.
The preparation of tablets: 20 tablets VB 6 (Nanjing Baijingyu Pharmaceutical Co., Ltd.) were taken to grind a fine powder.Next, a suitable amount powder (about one tablet mount) was taken to be dissolved with twicequartz-distilled water, and it was then transferred to 50 mL volumetric flask, diluted to the mark with twicequartz-distilled water.Final, it was filtered to remove infusible substance.
Electrochemical measurement: 5.0 mL of 0.10 mol•L -1 PBS containing a suitable amount of VB 6 was added to the 10.0 mL cell.Before experiment, the solution was purged with nitrogen for 10 minutes, and the flow of nitrogen was maintained during the experiment.In DPV measurements, the experiment parameters were following: initial potential: 0.3 V; final potential: 0.9 V; amplitude: 0.05 V; pulse width: 0.05 s; sample width: 0.0167.

SEM of the Different Modified Electrodes
Figure 1 displayed SEM morphologies of MWCNTs/ GCE (a) and Au NPs/MWCNTs/GCE (b).It was observed that the MWCNTs distributed uniformly on the surface of GCE and Au NPs had been uniformly decorated onto the surface of MWCNTs (Figure 1(b)).The diameter of Au NPs was about 50 nm.

Electrochemical Behaviors of VB 6 at Bare or Modified Electrode
Electrochemical behaviors of VB 6 at various modified electrode in 0.10 mol•L -1 PBS were investigated by CV technique.

Optimization of the Experimental Conditions
modified on GCE (Figure 2(c)), the response of VB 6 was enhanced; especially, the current intensity of VB 6 was the highest at Au NPs/MWCNTs modified electrode (Figure 2(d)).These facts might attribute to the synergistic effect of Au NPs and MWCNTs.At the same time, there was only an oxidation peak at +0.62 V and no reduction peak could be observed in CVs, indicting that the electrode reaction of VB 6 at the Au NPs/MWCNTs/GCE was irreversible.

The Effect of Deposition Time
In this work, Au NPs deposition time was optimized by CV technique.The results were shown in Figure 3.As can be seen, the current intensity increased rapidly with the augment of the deposition time increased from 5 to 20 s.The maximum current intensity was observed when the deposition time was 20 s.However, when the deposition times increased continuously, the current intensity decreased gradually.Therefore, 20 s was chosen as deposition time.
The effect of the scan rate on peak current of VB 6 was investigated at the Au NPs/MWCNTs/GCE, and the current intensity was linearly increased with the square of the scan rate, the linear regression equation was: I(μA) = 1.183 + 109.1υ1/2 (v/s) (r = 0.9992), indicating the electrode reaction of VB 6 at modified electrode was a diffusion control process.

The Effect of pH
The effect of the solution pH on the electrochemical response of VB 6 was investigated.The results were shown in Figure 4.As can be seen, the current intensity in-  creased with solution pH from 5.0 -7.0, then it decreased when the pH was over 7.0.The maximum current intensity was observed when pH was 7.0.The peak potential was linearly shifted negatively with the augment of solu-tion pH, indicating that protons had taken part in the electrode reaction process of VB 6 .Therefore, pH 7.0 was selected in all following experiments.

Calibration Curve
The calibration curve of VB 6 detection was performed with DPV technique.Under the optimized experimental conditions, a series of different concentrations of VB 6 were measured.The current intensity at + 0.62 V was used to obtain the calibration curve.The results showed that the current intensity was linear with the concentrations of VB 6 in the range of 1.59 to 102.74 μg•mL -1 as shown in Figure 5.The linear regression equation was I pa (μA) = 0.2759 c + 0.7007 (C-μg•mL -1 ) (r = 0.9994) with the detection limit of 0.53 μg•mL -1 (S/N = 3).

Stability and Reproducibility
The reproducibility of the modified electrode was investigated by successive measurements of 30 μg•mL -1 VB 6 by DPV (n = 6), the obtained peak current value was about the range of 8.79 to 9.24 μA with a relative standard deviation (RSD) of 2.05%.The stability of the modified electrode was investigated after it was storage in air for 2 weeks.The obtained peak currents were almost constant.Therefore, the modified electrode had a good stability and reproducibility.

Interference Study
In these experiments, we investigated the interference of several species on the determination of VB 6, such as VB 1 , VB 2 , AA, glucose, starch.The experiment results were shown in Table 1, from Table 1, there were no interferences existed for the determination of 30 μg•mL -1 VB 6 in the presence of 20-fold VB 1 , a 50-fold VB 2 , 100-fold AA, glucose and starch.

Analytical Applications
In this work, the injection and tablet of VB 6 were analyzed by the standard addition method.For comparison purposes, the obtained results were compared with that of China Pharmacopoeia Method [22] (shown in Tables 2, 3).From Table 2 and Table 3 we could observe that the obtained values were agreement with the results found by the China Pharmacopoeia Method.

Conclusions
The Au NPs/MWCNTs composite film modified electrode showed a strong electrochemical response towards VB 6 in pH 7.0 and AA showed no interference during VB 6 detection.The modified electrode had been applied in medication analysis, and obtained good results.

Figure 3 .
Figure 3.The plots of the peak currents of VB 6 vs electrodeposition time of Au NPs, c = 30 μg•mL -1 .

Table 1 . The inference of coexistent substances.
. the average peak current of VB 6 .b. the average peak current of VB 6 after adding different coexisting substances. a

Table 2 . Results for the determination of VB 6 in tablets.
The tables were obtained from Nanjing Baijinhyu Pharmaceutical Co., Ltd.

Table 3 . Results for the determination of VB 6 in injections.
The injections were obtained from Henan Topfond Pharmaceutical Co., Ltd.