The prevalence of chronic renal disease exceeds 10% in industrialized societies [1]. Oxidative damage is thought to be one of the main mechanisms involved in nearly all chronic renal pathologies. Both synthetic and natural antioxidants have been reported to be effective in preventing and curing such disease [2]. However, emerging information on the possible adverse effects of synthetic additives on food quality and human health has led to growing interest in natural antioxidants [3].
Salvia miltiorrhiza (Dan Shen) is a widely used herbal medicine (HM) in China, Japan, North America, and Europe [4]. It has been used for the treatment of cardiovascular diseases, including coronary heart disease [5], hyperlipidemia, and cerebrovascular diseases [6-10], because of its ability to prevent oxidative damage to cells, lipids, and DNA [3, 5-9, 11, 12]. Subsequently, evaluating the antioxidant properties of such medicines is critically important. Wei et al. [13] developed an HPLC-UV method to determine four phenolic acids in a Dan Shen formulation. However, UV detection has limited sensitivity and is highly prone to interference owing to its inability to distinguish between antioxidants and non-antioxidants. Chang and co-workers [11] used HPLC-chemiluminescence (CL) detection to obtain the antioxidant-activity-integrated fingerprint of Dan Shen as well as its total antioxidant capacity. This method was highly selective for components with antioxidant properties. However, the HPLC-CL method involved the use of a 30% (v/v) H2O2 reagent, which is thermodynamically unstable, especially at higher pH, and can be toxic. Electrochemical detection (ECD) offers higher sensitivity and better selectivity than optical techniques. Moreover, it is much safer than the abovementioned HPLC-CL method, since no toxic reagent is needed. Since the 1980s, there have been many publications describing the use of ECD to detect antioxidants [14, 15], such as phenolic compounds. However, most reported HPLC-ECD methods have used isocratic elution [16-20]. To analyze complex samples, such as HMs, gradient elution is much more useful owing to a higher peak capacity and better resolution [21]. Zheng and co-workers [22] developed a gradient HPLC-ECD method for determining phenolic compounds from Guanxintong tablets. This method was more sensitive than UV methods, but its sensitivity was still limited for lower concentration samples, and its linear range was only one to two orders of magnitude.
In this study, a new ECD, ECD 3000RS [23], with an amperometric cell was used to develop a gradient elution method for evaluating antioxidant properties of Dan Shen. Chromatographic conditions, including pH, buffer type and concentration, flow rate, water-acetonitrile gradient profile, and detection potential, were systematically investigated. Finally, the optimized method was utilized to quantify five phenolic compounds and to simultaneously obtain antioxidant fingerprint profiles and measure total antioxidant capacity of Dan Shen samples.
The chromatographic system consisted of a DGP-3600RS pump, a WPS-3000RS autosampler, and an ECD 3000RS detector with a column oven. An amperometric cell with a glassy carbon (GC) working electrode and palladium/hydrogen reference electrode was used for detection. A guard cell (Omni cell) was used before the injector to oxidize electro-active impurities in the mobile phase. Cyclic voltammograms of phosphate and acetate were obtained with an ICS 5000. Data were collected and analyzed by Chromeleon version 7.2. All of the abovementioned instrumentation and workstations were obtained from Thermo Fisher (Sunnyvale, CA, USA). The C18HC column (150 mm×2.1 mm, 5 μm) for phenolic analysis was obtained from Dalian Institute of Chemical Physics, Chinese Academy of Sciences [24]. Reference compounds of salvianolic acid B, rosmarinic acid, caffeic acid, lithospermic acid, and isoferulic acid were obtained from the National Institutes for Food and Drug Control (Beijing, China). HPLC grade acetonitrile (ACN), sodium dihydrogen phosphate (NaH2PO4) (98%), sodium acetate (NaOAc), acetic acid, and phosphoric acid (H3PO4) (85%-90%) were obtained from Thermo Fisher (Sunnyvale, CA, USA). Water was prepared using a Barnstead GenPure system (Sunnyvale, CA, USA).
The mobile phases were A: ACN, B: 100 mmol/L NaH2PO4 (pH 2.8) aqueous, and C: water. The column temperature was 30 ℃. The gradient was changed from 10%A, 20%B, and 70%C to 35%A, 20%B, and 45%C over 30 min. The flow rate was 0.2 mL/min. The ECD cell potentials were 800 and 700 mV for the amperometric and Omni cells, respectively. Data were collected at 10 Hz. The injection volume was 10 μL. The Omni cell was used before the injector to oxidize electro-active impurities in the mobile phase and thus obtain a lower noise baseline for detection. Dan Shen samples were obtained from Shandong and Sichuan (China) and authenticated by Xiaoping Yang from Dalian Institute of Chemical Physics, Chinese Academy of Sciences.
Fourteen batches of Dan Shen were investigated. Samples were powdered to a homogeneous size and extracted [25]. Twenty milligrams of each sample powder was weighed accurately and placed in an Erlenmeyer flask before adding 200 mL of methanol-water (70:30, v/v). The mixture was shaken and sonicated for 2 h. After filtration to remove particulates, 1.0 mL of extract was diluted with 39 mL of phosphate buffer (pH 2.8), and then centrifuged at 10 000 r/min for 10 min. The supernatants were filtered through a 0.22 μm membrane to obtain solution 1, and then analyzed by HPLC.
The linearities of caffeic acid, isoferulic acid, rosmarinic acid, lithospermic acid, and salvianolic acid B were studied using at least five concentration levels over the range of 1.6-1 591.0, 1.2-1 167.0, 1.8-890.0, 2.6-1 310.0, and 3.6-900.0 μg/L, respectively. Intra-day and inter-day repeatabilities were studied at low, moderate, and high mass concentration levels. The low mass concentrations were 49.7, 36.5, 27.8, 40.9, and 28.1 μg/L, the moderate mass concentrations were 198.9, 145.9, 111.2, 163.8, and 112.5 μg/L, and the high mass concentrations were 795.5, 583.5, 445.0, 655.0, and 450.0 μg/L for caffeic acid, isoferulic acid, rosmarinic acid, lithospermic acid, and salvianolic acid B, respectively. The recoveries of targeted compounds in Dan Shen samples were determined by the following equation:
Recovery=(experimental mass concentration/theoretical mass concentration)×100%.
Five phenolic compounds, including rosmarinic acid, lithospermic acid, salvianolic acid B, isoferulic acid, and caffeic acid (Supporting Information 1, http://www.chrom-China.com/UserFiles/File/sp1703038_Supporting_Information.pdf) were chosen to optimize the separation and detection conditions, as they are highly bioactive compounds reported to be present in many types of HMs including Dan Shen.
Both methanol and ACN are widely used in HPLC separations. ACN is more commonly used for ECD since methanol can be oxidized by some electrodes under certain conditions [26]. To avoid background interference, ACN was used in this study.
Isocratic elution (15% (v/v) ACN dissolved in 20 mmol/L NaH2PO4 (pH 2.8) aqueous) was initially used for the separation of Dan Shen extracts, since it is compatible with all ECD detectors. However, long run times ( > 30 min) were needed, and broad, flattened peaks were observed for some of the late-eluting analytes. More importantly, some hydrophobic components present in the Dan Shen extractions could not be eluted using these isocratic conditions (data not shown). Compared with isocratic elution, gradient elution is much more widely used for the analysis of HMs owing to a higher peak capacity and better resolution [27]. Since ECD 3000 RS is compatible with gradient elution [23], a gradient method was optimized in this study. Preliminary results with a linear gradient (0-30 min, 10%-70% (v/v) ACN) demonstrated poor resolution and weak retention for the five targeted analytes (data not shown). Hence, the ACN gradient slope was reduced from 2%/min to 0.83%/min, and the best resolution was obtained with the following conditions: 0-30 min; 10%-35% (v/v) ACN. This gradient program was successfully applied to the analysis of real samples and reference compound mixtures (Fig. 1).
Both acetate and phosphate buffers are widely used with ECD. To aid in the selection of HPLC buffer, cyclic voltammetry with a GC electrode was used to characterize acetate and phosphate buffers in the potential range of-1 000 mV to + 1 000 mV (Supporting Information 2, http://www.chrom-China.com/UserFiles/File/sp1703038_Supporting_Information.pdf). Phosphate was found to generate lower currents than acetate at potentials > +500 mV, which agrees well with previous reports [28-30] that acetate can be oxidized on Pt and GC electrodes at high potentials. Since lower currents are typically associated with lower baseline noise, it is expected that phosphate would provide lower chromatographic baseline current and noise than acetate at positive detection potentials. This was confirmed as shown in Fig. 2a. In addition, less baseline drift was observed with phosphate during aqueous ACN gradients. Therefore, phosphate was chosen for this method.
Baselines obtained with 20 mmol/L and 30 mmol/L buffer are compared in Fig. 2a. No obvious increase in baseline noise or chromatographic drift was observed when the buffer concentration increased. This is partly attributed to the use of an Omni cell, which can reduce the adverse effect of electro-active impurities in the mobile phase by oxidizing them. In addition, both 20 mmol/L and 30 mmol/L phosphate (pH 2.8) buffer provided symmetrical peaks for all targeted compounds (Supporting Information 3, http://www.chrom-China.com/UserFiles/File/sp1703038_Supporting_Information.pdf). Since a greater concentrated buffer is more prone to precipitate in an HPLC system, 20 mmol/L phosphate was used as mobile phase additive for the separation of phenolic compounds.
The effect of mobile phase pH on the retention of phenolic compounds primarily depends on the pKa of its functional groups. As shown in Supporting Information 3, longer retention and better peak shape was obtained using a more acidic mobile phase. For example, the asymmetry factor (As) of salvianolic acid B was 1.02 at pH 2.8 and 0.61 at pH 4.8. The higher pH increases the extent of ionization of both the column's silanol groups [31] and the phenolic analytes. Moreover, at high pH the analytes are more hydrophilic. Thus, the analyzed compounds are less retained at high pH. Because of heterogeneous transfers (hydrophobic interaction and repulsive interaction), peak asymmetry was also observed with phenolic compounds. This agrees well with the study of Guiochon et al. [32-34]. At pH 2.8, increasing the buffer concentration had no obvious effect on retention behavior since ionization of phenol groups is inhibited thus minimizing any electrostatic interactions. Higher ECD sensitivity can be obtained for phenolic compounds by using a lower pH mobile phase, as it provides lower current [35], higher response [14], and sharper peaks. Thus, a pH 2.8 phosphate buffer was used for further separation of phenolic compounds.
The effect of flow rate was investigated in the range of 0.2 to 1.0 mL/min. For UV detection (280 nm), flow rate had no obvious effect on background response (data not shown). For ECD, however, the background response increased with increasing flow rate (0.2-0.7 mL/min) (Fig. 2b) and was relatively constant above 0.7 mL/min. That is, a lower background response was obtained with a lower flow rate for amperometric detection. According to a previous study [36], the amperometric signal (I) is related to the instantaneous flow rate (νf) and concentration (C) existent in the detector as represented by the equation: I=nFAkνfαC, where k and a are constants related to the diffusion coefficient and geometric parameters. The items of n, F and A are number of electrons, Faraday constant and the area of flow cell. Thus, a higher amperometric signal can be obtained with a lower flow rate. Since a lower flow rate can provide a lower current and higher response, a flow rate of 0.2 mL/min was used to separate phenolic compounds. The effect of longitudinal diffusion was not serious at a flow rate of 0.2 mL/min, since the average width (50%) (0.174 min) and asymmetry factor (1.01) were good enough. Lower flow rates were not investigated because the effects of diffusion coefficients, gradient delay time, and longitudinal diffusion were expected to be significantly increased.
The last optimization step involved investigating the effect of amperometric cell potential. Signal-to-noise ratios (S/N) of five phenolic compounds were studied over the working electrode potential range of +200 to +800 mV. As shown in Fig. 3, at pH 2.8, all investigated phenolic compounds had a low response at low potential (+200 or +300 mV), and there was no response for isoferulic acid until the potential was increased to +400 mV. The highest S/N for all target compounds was obtained at +700 mV and thus used for further study.
The sensitivity of the target compounds was determined by evaluating the chromatograms at different compound concentrations. The limit of quantification (LOQ) was taken as the sample concentration at which peaks could be integrated properly with an S/N of 10. Table 1 shows that the LOQ values obtained with ECD were much lower than those obtained with UV [37], demonstrating the higher sensitivity of ECD for these compounds. For example, the LOQ of salvianolic acid B achieved with ECD (3.55 μg/L) was about 31 times lower than with UV (112.5 μg/L). The current ECD method also provides much higher sensitivity than a previously published ECD method [14]. For example, the LOQ obtained for salvianolic acid B with the current ECD method (3.55 μg/L) is about one-sixth that of the previous study (23.31 μg/L) [14]. As key bioactive substances often exist at low concentration [38], the developed ECD method has great potential for discovering new biomarkers, especially antioxidants.
Linearity was assessed from duplicate 10 μL injections of ≥5 levels of each compound over the concentration ranges described in Section 1.4. Coefficients of determination from linear least-squares regression analysis of peak area versus mass concentration were between 0.999 2 and 1.000 0 as shown in Table 1. Compared with a previous method [22], which provided a linear range of 1 to 2 orders of magnitude, the current ECD method provides a much wider linear response range (9-1 500 μg/L). This demonstrates that the described ECD method can be used to determine both low and high concentrations of phenolic acids. Accuracy of the linear equation was evaluated based on the recoveries of target compounds at four mass concentrations and calculated as recovery=(Ctheoretical-Cmeasured)/Ctheoretical×100%. Cmeasured was the mass concentration determined from the linear calibration equation, and Ctheoretical was the mass concentration of the standard solution. For the concentration span from 50 to 800 μg/L, the experimental values were all within ±5% of theoretical values (Table 2). Therefore, the accuracy of the current method was found to be acceptable for the determination of phenolic compounds with a wide mass concentration range.
Repeatability and intermediate precision were investigated at three concentration levels as described in Section 1.4, and results are shown in Table 1. For moderate and high levels, intra-day RSDs for all target compounds were < 2.0% while that for the low level were < 3.0%. The inter-day RSDs of all compounds were not more than 4.01%. All of the above results demonstrate that the current method had good precision.
To evaluate the method's applicability, we carried out an evaluation with 14 batches of Dan Shen samples obtained from different places. Since UV was recommended by the Chinese Pharmacopeia 2015 (CP2015) for the quantification of Dan Shen, the UV chromatogram of Dan Shen was recorded and compared with the ECD chromatogram acquired in this study.
Quantitative studies of phenolic compounds from 14 batches of Dan Shen samples were conducted using the HPLC-ECD method developed in Sections 2.1 and 2.2 (Fig. 4). For these studies, two 20 milligram portions of each Dan Shen sample were separately carried through the sample pretreatment steps described in Section 1.3, and each extract was analyzed in duplicate. Using linear calibration, the amounts of these five phenolic compounds were determined, and results are listed in Table 3. In all samples, the amount of the target compounds increased in this order: caffeic acid < isoferulic acid < rosmarinic acid (lithospermic acid) < < salvianolic acid B. Samples numbered 1, 2, and 3 had a lower concentration of lithospermic acid than other samples, which indicated that these three samples were different from the others. The values determined by ECD were slightly lower than those determined by UV (Table 3). As shown in Fig. 4, it is clear that some peaks (*) in the UV chromatogram were not detected by ECD. A main consideration in this regard is that these peaks (*) are less likely to be antioxidants since they were not as easily oxidized electrochemically as the target phenolic compounds. UV is less selective and therefore many non-antioxidants can also be detected at 286 nm. Thus, there are fewer interferences from co-eluted non-antioxidants in an ECD chromatogram than in a UV chromatogram. Taking the abovementioned factors into consideration, ECD was found to be an effective alternative to UV for quality evaluation of Dan Shen.
The recoveries of caffeic acid, isoferulic acid, rosmarinic acid, lithospermic acid, and salvianolic acid B from these 14 batches of Dan Shen samples are presented in Table 4. Recoveries for all target compounds in each sample were in the range of 96.2% to 105.5%. This demonstrates that the described method can be successfully used to determine these five phenolic compounds in Dan Shen samples.
An HPLC-ECD method was optimized for determining antioxidants from Dan Shen. The effects of mobile phase pH, flow rate, organic solvent type and gradient steepness, and buffer type and concentration on peak shape and sensitivity of ECD were systematically investigated. The optimized HPLC-ECD method provided good recovery and intra-day (RSDs≤2.10%) and inter-day (RSDs≤4.01%) repeatabilities. Compared with reported ECD methods, the developed method offered much higher sensitivity and a wider linear range (a dynamic range of up to 104 for determination of five target compounds). Compared with UV, the developed ECD method was found to be 16 to 31 times more sensitive and had fewer interferences from co-eluting non-antioxidants.
2017,
Vol. 35 Issue (8): 897-905
PDF
2017,
Vol. 35 Issue (8): 897-905
PDF