Highly-sensitive Detection of Salvianolic Acid B using Alumina Microfibers-modified Electrode

Abstract

Alumina microfibers with porous structures were prepared through hydrothermal reaction, and then used to modify the surface of carbon paste electrode (CPE). After modification with alumina microfibers, the electrochemical activity of CPE was found to be greatly improved. On the surface of alumina microfibers-modified CPE, the oxidation peak current of salvianolic acid B, a main bioactive compound in Danshen with anti-oxidative and anti-inflammatory effects, was remarkably increased compared with that on the bare CPE surface. The influences of pH value, amount of alumina microfibers and accumulation time were studied. Based on the strong signal amplification effects of alumina microfibers, a novel electrochemical method was developed for the detection of salvianolic acid B. The linear range was from 5 ${\mu}gL^{-1}$ to 0.3 mg $L^{-1}$, and the detection limit was 2 ${\mu}gL^{-1}$ (2.78 nM) after 1-min accumulation. The new method was successfully used to detect salvianolic acid B in ShuangDan oral liquid samples, and the recovery was over the range from 97.4% to 102.9%.

Introduction

Danshen (Salvia miltiorrhiza Bunge) is a well-known Chinese herbal medicine used more than one thousand of years in treating cardiovascular, hepatic and renal diseases.12 Salvianolic acid B, a kind of polyphenolic antioxidants, is the most abundant and bioactive member of Danshen.3 Studies have shown that salvianolic acid B can exert various antioxidative and anti-inflammatory activities in different disease models.45 Furthermore, salvianolic acid B has been found to possess many other pharmacological functions, such as attenuating brain damage,6 inhibitory effects on apoptosis,7 cardioprotective effects,8 and potential candidate for Alzheimer’s disease therapy.9 Apparently, developing simple, rapid and sensitive analytical method for salvianolic acid B is quite important and very interesting.

Up-to-date, the extensively-used method for the determination of salvianolic acid B was high-performance liquid chromatography (HPLC).10-12 Although HPLC possesses high sensitivity and excellent selectivity, it is time consuming and the operation is relatively complicated. Compared with HPLC, electrochemical detection exhibits some advantages such as short analysis time, low cost and good handling convenience. However, the study regarding electrochemical determination of salvianolic acid B is very limited.

Due to its unique properties, alumina microfibers were proven to be superior electrode materials and exhibited remarkable signal enhancement effects for amaranth,13 and sunset yellow.14 Herein, alumina microfibers in diameter of 100 nm were prepared and subsequently used to modify the CPE surface. The electrochemical responses of salvianolic acid B were investigated. Compared with the bare CPE, the alumina microfibers-modified CPE greatly increased the oxidation peak current of salvianolic acid B, showing strong signal amplification effects. Alumina microfibers with porous structures provided numerous active sites for the oxidation of salvianolic acid B. Undoubtedly, the accumulation efficiency of salvianolic acid B on electrode surface was greatly improved, resulting in obvious signal enhancement. After optimizing analytical parameters, a novel, simple, rapid, sensitive and reliable electrochemical method was developed for the detection of salvianolic acid B.

 

Experimental Section

Reagents. All chemicals were of analytical grade and used as received. Salvianolic acid B (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) was dissolved into ethanol to prepare 0.1 mg mL−1 standard solution, and stored in the fridge at 4 ℃. Urea, aluminium nitrate, graphite powder (spectral reagent) and paraffin oil were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). Doubly distilled water was used throughout.

Instruments. Electrochemical measurements were performed on a CHI 830C electrochemical workstation (Chenhua Instrument, Shanghai, China). A conventional three-electrode system, consisting of a alumina microfibers-modified carbon paste working electrode, a saturated calomel reference electrode (SCE) and a platinum wire auxiliary electrode, was employed. Transmission electron microscopy (TEM) images were taken on a Tecnai G220 microscope (FEI Company, Netherlands).

Preparation of Alumina Microfibers. In a typical synthesis, Al(NO3)3·9H2O was firstly dissolved in doubly distilled water to form a clear solution and then urea was added. The molar ratio of Al:Urea:H2O was 1:9:90. After being totally dissolved, the mixture was transferred to a Teflon-lined stainless-steel autoclave, and reacted at 100 ℃ for 12 h. Finally, the solid was filtered off, washed with doubly distilled water, dried at 80 ℃ for 24 h, and calcined at 500 ℃ for 2 h.

Electrode Modification. The resulting alumina microfibers (0.15 g) and graphite powder (0.85 g) were exactly weighed, and put in a carnelian mortar. The total mass was controlled at 1.0 g and the mass content of alumina microfibers was 15%. After that, 0.30 mL paraffin oil was added into the powder and then mixed homogeneously. Finally, the resulting carbon paste was tightly pressed into the end cavity of electrode body, and the electrode surface was polished on a smooth paper. The unmodified CPE was also prepared without addition of the prepared alumina microfibers.

Analytical Procedure. Unless otherwise stated, 0.1 M acetate buffer with pH of 4 was used as supporting electrolyte for the detection of salvianolic acid B. After 1-min accumulation at 0.1 V, the differential pulse voltammograms were recorded from 0.1 to 0.6 V, and the oxidation peak current at 0.32 V was measured as the analytical signal for salvianolic acid B. The pulse amplitude was 50 mV, the pulse width was 40 ms and the scan rate was 40 mV s−1.

 

Results and Discussion

Characterization of Alumina Microfibers. The prepared alumina samples were characterized using TEM, and the image was shown in Figure 1. It was found that the obtained alumina samples consisted of regular fibers. The diameter was over the ranger from 90 to 110 nm, and the length was about 450-550 nm. Moreover, porous structures were clearly observed.

Figure 1.TEM images of alumina microfibers.

Figure 2.DPV curves of CPE (a, b) and alumina microfibers-modified CPE (c, d) in pH 4 acetate buffer (a, c) and in the presence of 50 mg L−1 salvianolic acid B (b, d). Accumulation was performed at 0.1 V for 1 min.

Signal Amplification of Alumina Microfibers-modified CPE. The oxidation responses of salvianolic acid B on CPE and alumina microfibers-modified CPE were compared using differential pulse voltammetry (DPV). In pH 4 acetate buffer containing 50 μg L−1 salvianolic acid B, an oxidation peak was observed on CPE surface after 1-min accumulation at 0.1 V (Fig. 2(b)). The peak potential was 0.32 V and the peak current was low, suggesting that the oxidation activity of salvianolic acid B is poor on the bare CPE surface. However, the oxidation signal was greatly improved on the surface of alumina microfibers-modified CPE (Fig. 2(d)). The notable peak current enlargement indicates that alumina microfibers exhibit strong signal amplification effects toward the oxidation of salvianolic acid B. From TEM measurement, we knew that the prepared alumina consisted of regular and porous microfibers, providing high accumulation efficiency for salvianolic acid B. Therefore, the surface concentration of salvianolic acid B was significantly improved on alumina microfibers-modified CPE, resulting in considerable oxidation signal enlargement. Besides, the DPV curves on CPE (Fig. 2(a)) and alumina microfibers-modified CPE (Fig. 2(c)) in the absence of salvianolic acid B were virtually featureless, revealing that the oxidation wave at 0.32 V was due to the oxidation of salvianolic acid B. In conclusion, the comparison of Figure 2 clearly demonstrates that the alumina microfibers surface is more active for the oxidation and detection of salvianolic acid B.

Electrochemical Detection of Salvianolic Acid B. The electrochemical behaviors of salvianolic acid B in different solutions were examined using cyclic voltammetry (CV) to choose the suitable supporting electrolyte. Figure 3 shows the CV responses of 1 mg L−1 salvianolic acid B on alumina microfibers-modified CPE in 0.1 M acetate buffer solutions with pH values of 3.6, 4, 4.6, 5 and 5.6. A pair of redox peaks were observed and the peak potentials were found to negatively shift with the increase of pH values. As to oxidation peak, the peak potential shifted linearly with pH, and the slope of Epa-pH plot was −54.2 mV/pH, suggesting that the number of transferred proton and electron was the same in the oxidation of salvianolic acid B. In addition, the oxidation peak currents of salvianolic acid B gradually increased when changing pH value from 5.6 to 4, and then changed very slightly when further decreasing pH value to 3.6. To achieve high response signal, pH 4 acetate buffer was employed for the detection of salvianolic acid B.

Figure 3.CV curves of 1 mg L−1 salvianolic acid B on alumina microfibers-modified CPE in 0.1 M acetate buffer with different pH values. Scan rate: 100 mV s−1.

Figure 4.DPV curves of 50 mg L−1 salvianolic acid B on alumina microfibers-modified CPE from different starting potentials: −0.3 V (a), −0.2 V (b), −0.1 V (c), 0 V (d), 0.1 V (e) and 0.2 V (f). Accumulation time: 1 min.

For handling convenience and further enhancing the sensitivity for salvianolic acid B detection, accumulation was employed under the initial potentials. Figure 4 depicts the DPV curves of salvianolic acid B from different starting potentials using alumina microfibers-modified CPE. As changing the accumulation potential from −0.3 to 0.1 V, the oxidation signal of salvianolic acid B obviously increased, indicating that accumulation of salvianolic acid B on alumina microfibers surface is higher at positive potential. When further moving the starting potential from 0.1 to 0.2 V, the oxidation peak current of salvianolic acid B changed slightly. To achieve high sensitivity and excellent oxidation shape, the accumulation was performed at 0.1 V, and the DPV sweep also started from 0.1 V.

Figure 5.Influence of mass content of alumina microfibers on the oxidation peak current of 50 mg L−1 salvianolic acid B. Accumulation was conducted at 0.1 V for 1 min. Error bar represents the standard deviation of triple measurements.

Figure 5 displays the effect of mass content of alumina microfibers on the oxidation peak current of salvianolic acid B. By extending the mass content from 0 to 15%, the peak current of salvianolic acid B enhanced greatly. This reveals that alumina microfibers greatly enhance the surface activity of CPE. As further improving the mass content to 20%, no obvious peak current enlargement was noticed for salvianolic acid B, suggesting a saturated state of alumina microfibers on CPE surface. However, the oxidation peak current of salvianolic acid B decreased when further increasing the mass content to 25%. This may be due to the fact that too much alumina microfibers lower the conductivity and block the electron transfer of salvianolic acid B. In this work, the optimal content of alumina microfibers was selected as 15%.

Figure 6.Oxidation peak current of 50 mg L−1 salvianolic acid B as a function of accumulation time. Error bar represents the standard deviation of triple measurements.

The influence of accumulation time on the oxidation signal of salvianolic acid B was studied. As seen in Figure 6, the oxidation peak currents of salvianolic acid B on alumina microfibers-modified CPE remarkably increased with accumulation time from 0 to 1 min. The notable signal enhancement indicates that accumulation is effective to improve the detection sensitivity. Longer accumulation time than 1 min did not significantly enhance the oxidation peak current of salvianolic acid B, suggesting that the amount of salvianolic acid B approached a limiting value. Considering sensitivity and efficiency, 1-min accumulation was employed.

The successive measurements using one same alumina microfibers-modified CPE were examined. Unfortunately, the oxidation signal of salvianolic acid B decreased continuously. The strong surface sorption and fouling were the main reason. Thus, alumina microfibers-modified CPE was used for single detection, and the reproducibility between multiple electrodes was evaluated by parallel determining the oxidation peak current of 50 μg L−1 salvianolic acid B. The relative standard deviation (RSD) is 4.1% for eleven alumina microfibers-modified CPEs, indicative of excellent fabrication reproducibility and detection precision.

The potential interferences for the detection of salvianolic acid B were studied using DPV. The oxidation peak current of salvianolic acid B was individually measured in the presence of different concentrations of interferents, and then the peak current change was checked. No influence on the determination of 50 μg L−1 salvianolic acid B was found after the addition of 0.5 mg L−1 glucose, glycine, cystine and tryptophan; 50 mg L−1 ascorbic acid, uric acid, xanthine, hypoxanthine and protocatechuic aldehyde; 5 mg L−1 of paeonol, salvianolic acid A, protocatechuic acid, brucine, gallic acid and danshensu (peak current change < 10%).

Under the optimized conditions, the linear range and detection limit was investigated using DPV. It was found that the oxidation peak current of salvianolic acid B (𝑖p, μA) was proportional to its concentration (C, μg L−1) over the range from 5 μg L−1 to 0.3 mg L−1, obeying the following equation: 𝑖p = 0.0507 C. The correlation coefficient (R) was 0.997, indicative of good linearity. Additionally, the detection limit was evaluated to be 2 μg L−1 based on three signal to noise ratio.

Table 1.Detection of salvianolic acid B in Shuang Dan oral liquid samples

Analytical Application. In order to assess the performance of the proposed method in real sample analysis, it was used to detect the content of salvianolic acid B in a traditional Chinese medicinal preparation ShuangDan oral liquid. The samples were purchased from a local pharmacy and diluted by 1000-fold with doubly distilled water. After adding 20 μL diluted sample solution into 10.0 mL pH 4 acetate buffer, the DPV curve was recorded after 1-min accumulation, and the peak current was measured at 0.32 V. Each sample was determined by five parallel detections, and the RSD was below 5%, revealing excellent precision. The content of salvianolic acid B was obtained by the standard addition method, and the results were listed in Table 1. In addition, the recovery of salvianolic acid B was also performed to testify the accuracy of this method. The value of recovery was over the range from 97.4% to 102.9%, suggesting that this method is effective and reliable for the detection of salvianolic acid B.

 

Conclusion

Modification with alumina microfibers greatly enhanced the surface activity of CPE due to regular fiber-like and porous structures. Alumina microfibers exhibited high accumulation efficiency to salvianolic acid B and significantly enhanced the oxidation signal of salvianolic acid B. As a result, a novel electrochemical method was developed for salvianolic acid B detection. This new method possessed high sensitivity, rapid response, good reproducibility, excellent accuracy and promising application.