Infrared-assisted extraction followed by high performance liquid chromatography to determine angoroside C, cinnamic acid, and harpagoside content in Scrophularia ningpoensis

The dried roots of S. ningpoensis were purchased from the Anhui Dechang Pharmaceutical Co., Ltd. (Anhui, China) and ground to a fine powder. Angoroside C (analytical grade; lot no. 151205, purity ≥98%) (Fig. 1a) was obtained from Shanghai Kangbiao Chemicals Co., Ltd. (Shanghai, China). Cinnamic acid (analytical grade; lot no. 111730–200,604) (Fig. 1b) and harpagoside (analytical grade, lot no. 110786–200,503) (Fig. 1c) were purchased from the National Institute for the Control of Pharmaceuticals and Biological Products (Beijing, China). Acetic acid was obtained from Revitalization of Chinese Chemical Plant (Jiangsu, China). Methanol (HPLC grade) was purchased from Merck (New Jersey, USA). Deionized water was purified using an Auto ScienceAP-01P System from Tianjin Automatic Science Instrument Co., Ltd. (Shanghai, China). The infrared lamp (275 W) was obtained from Shanghai Tour Light Electrical Appliance Co. Ltd. (Shanghai, China).

The apparatus for IRAE, according to our previously reported method, is illustrated in Fig. 2 [28]. During the extraction process, the conduit for cooling water was connected to the condenser to prevent solvent evaporation.

According to our previously reported method [1, 28], 1 g of S. ningpoensis dried root was placed in a 100-mL flask with 25 mL of 37.5% ethanol-distilled solvent (the optimum extraction solvent obtained using the IRAE method). The flask with the sample was placed in an MO-2270 M1 model microwave oven and heated at 400 W for 4 min. Simultaneously, a condenser with a continuous flow of cooling water was connected to condense the solvent vapor.

According to our previously reported method [28], 1 g of S. ningpoensis dried root was transferred into a 100-mL flask containing 25 mL of 37.5% ethanol-distilled solvent (the optimum extraction solvent obtained using the IRAE method). The irradiation time was 60 min.

Stock solutions (1 mg/mL) of angoroside C, cinnamic acid, and harpagoside were prepared by dissolving them in methanol. Working standard solutions of concentrations 5, 10, 25, 50, 100, and 200 μg/mL for angoroside C and harpagoside, 2.5, 5, 12.5, 25, 50, and 100 μg/mL for cinnamic acid were prepared by diluting the respective stock solutions with methanol:water (50:50). The samples were stored at 4 °C. The calibration curves were obtained by weighted linear regression (weighing factor 1/x); the peak area was plotted versus the analyte concentration.

An Agilent (Palo Alto, CA, USA) 1100 LC system, equipped with a G1311A Quatpump, a G1322A vacuum degasser, a G1316A Thermostatted Column Compartment, a G1314A variable wavelength UV-visible detector, and an HP 1100 series manual injector with a 20-μL fixed loop, was used for the analysis. The detector was operated at 278 nm and peak areas were integrated automatically using Hewlett–Packard ChemStation software program (Rev. A. 10. 02 [1757]).

An Agilent TC C18 column (200 mm × 4.6 mm i.d., 5 μm particle size) was used to separate the analytes. The mobile phase consisted of solvents A (0.2% acetic acid) and B (methanol). The gradient elution steps were from 65:35 to 40:60 (A:B) over 15 min with a flow rate of 1 mL/min and column temperature of 25 °C. After each run, the column was re-equilibrated for 5 min. Peak areas were used for quantification.

To validate the method, the linearity, detection limit, repeatability, accuracy, and recovery were evaluated. The intra- and inter-day precision was determined by analyzing calibration samples during a single day and on three consecutive days, respectively. The relative standard deviation (RSD, %) was calculated based on the obtained peak area.

Solutions prepared using S. ningpoensis sample (n = 6) were used to analyze the reproducibility of the method. The accuracy of this method was evaluated using a recovery test. Accurate amounts of the reference compounds were transferred to S. ningpoensis sample, and then extracted and analyzed using the developed method. The recovery was calculated using the following formula: recovery (%) = (amount found-original amount) / amount added × 100 (n = 6).

Figure 4 shows the representative chromatograms of the angoroside C, cinnamic acid, and harpagoside (equivalent to 25 μg/mL) standards, and S. ningpoensis sample. The resolution values, symmetry, and theoretical plates of angoroside C, cinnamic acid, and harpagoside were above 3, 0.8, and 5000, respectively. The retention time of angoroside C, cinnamic acid, and harpagoside was 10.7, 13.5, and 17.9 min, respectively.

The linearity range of angoroside C, cinnamic acid, and harpagoside was 5–200 (5, 10, 25, 50, 100, and 200 μg/mL), 2.5–100 μg/mL (2.5, 5, 12.5, 25, 50, and 100 μg/mL), and 5–200 (5, 10, 25, 50, 100, and 200 μg/mL), respectively.

The calibration curves (Additional file 1: Figure S1) of angoroside C, cinnamic acid, and harpagoside were as follows: y = 21.86 x-7.66 (r = 0.9998), y = 152.60 x + 10.41 (r = 0.9998), and y = 44.99x-11.97 (r = 0.9997), respectively (n = 3; y: peak area; x: concentration, μg/mL).

All the RSD values of intra- and inter-day precision (Additional file 2: Figure S2, Additional file 3: Figure S3, Additional file 4: Figure S4), reproducibility, and recovery (Additional file 5: Figure S5) were less than 3% (Tables 1 and 2). The LOQ (Additional file 6: Figure S6) of angoroside C, cinnamic acid, and harpagoside was 2.5, 1, and 1.5 μg/mL, respectively (S/N = 10), which were considerably lower than the concentration in S. ningpoensis. The results demonstrated that the developed method is sensitive enough to analyze angoroside C, cinnamic acid, and harpagoside in S. ningpoensis.

The stability value of angoroside C, cinnamic acid, and harpagoside was 1.29, 1.67, and 0.63%, determined by periodic analysis of the same sample (0, 2, 4, 8, and 24 h), respectively.

Figure 4b presents the HPLC chromatogram of angoroside C, cinnamic acid, and harpagoside in S. ningpoensis sample obtained by IRAE under the optimal conditions. According to the calibration curves, the concentration of angoroside C, cinnamic acid, and harpagoside in S. ningpoensis was calculated, and the analytical results of IREA, MAE, and USE are listed in Table 3. There was no significant difference in the respective concentrations between the samples obtained using the IRAE and MAE methods. The concentration of angoroside C, cinnamic acid, and harpagoside in S. ningpoensis sample obtained using the proposed method was significantly higher than the respective concentrations in the samples obtained using the USE method (p < 0.05).