Simultaneous characterization and quantification of flavonoids in Euonymus alatus (Thunb.) Siebold from different origins by HPLC-PAD–MS

2.1 Materials and chemicals

The herb samples were collected from ten different provinces in China (Shandong, Heilongjiang, Shanxi, Beijing, Yunnan, Hunan, Henan, Jilin, Jiangsu and Shanghai), and identified by Prof. Chun-Min Li (Heilongjiang Institute for Drug Control). The dried samples were crushed into powder and stored in an exsiccator. Different medicinal portions (leaf, fruit, stem, pterygium, and root) collected from Heilongjiang were also for comparative analysis. The standards of rutin (batch number: 100080–200306; MWs: 610) and quercetin (batch number: 100081–200406; MWs: 302) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Their structures are shown in Fig. 1. Methanol of chromatographic grade (TEDIA Company, USA) was used for the HPLC analysis. Deionized water was purified by a Milli-Q system (Millipore, Bedford, MA, USA). Ethanol and methanoic acid of A.R. grade were used for the plant extraction and mobile phase, respectively.

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Figure 1

The chemical structures of rutin (A) and quercetin (B).

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2.2 Apparatus and chromatographic conditions

A SHIMADZU HPLC system, consisting of a binary pump, an on-line degasser, and a PAD was used for acquiring chromatograms and UV spectra. For chromatographic analysis, a Shim C₁₈ (4.6 mm × 150 mm, 5 μm) column with Kromasil C₁₈ (4.6 mm × 10 mm, 5 μm) guard column was used at 30 °C. The mobile phase consisted of methanol–water–methanoic acid (42:57.5:0.5, v/v/v) and the flow rate was 1 mL min⁻¹. All solutions were filtered through a membrane filter (0.45 μm) prior to HPLC analysis and the injection volume was 10 μL. The compounds were monitored at 360 nm and UV spectra were recorded in the range 210–400 nm.

ESI–MS was performed by MICROMASS® Quattro Micro^TM API. The flow rate was 1 mL min⁻¹ with 14% of eluent being split into the inlet of the mass spectrometer. The mass spectra were recorded with ESI in the negative mode. The parameters were as follows: capillary voltage: 2.50 kV; cone voltage: 40 V; extractor voltage: 3 V; RF lens voltage: 0.3 V; source temperature: 100 °C; desolvation temperature: 200 °C; desolvation gas flow rate: 350 L h⁻¹; cone gas flow rate: 50 L h⁻¹; scanning range: from 100 to 1000 amu. These parameters were optimized in preliminary experiments to get the highest abundance of the targeted molecular-related ions. N₂ was used as both dry gas and nebulizer gas.

2.3 Stock and working standard solutions

Standard stock solutions of quercetin (0.08 mg mL⁻¹) and rutin (0.015 mg mL⁻¹) were prepared in 25 mL volumetric flasks and diluted with methanol to obtain appropriate concentrations for the establishment of calibration curves. The flasks were sealed by elastic plastic film (Parafilm, Chicago, IL, USA) and stored in a refrigerator (4 °C) until the analysis.

2.4 Sample preparation

Powdered samples (100 mesh, 2.5 g) were extracted three times with 25 mL of 60% (v/v) ethanol with reflux for 1.5 h. The extracts were filtered under vacuum and condensed into 20 mL. After resting for 12 h (4 °C), the concentrated solutions were filtrated under vacuum to remove chlorophyll. The extracts were dried to produce yellow powder, which were then mixed with 200 mL methanol.

2.5 Method validation

The proposed chromatographic method was validated to determine the linearity, limit of detection (LOD), precision, repeatability, stability and accuracy.

The calibration curve for each compound was established with six different concentrations by plotting the peak area versus concentration. Linear regression analysis was performed by the external standard method.

The LOD was defined as the lowest concentration of analytes in a sample that can be detected and was determined on the basis of the signal-to-noise ratios of 3:1. The standard solutions of the compounds for LOD were prepared by diluting them sequentially.

The precision, repeatability and stability of the method were assessed in this procedure. The precision test was carried out by injecting the same sample solution six times. The repeatability was assessed by analyzing six individual prepared samples. Stability was tested by analyzing one sample solution stored at room temperature for 0, 2, 4, 8, 16, and 24 h after preparation. The peak areas of rutin and quercetin were recorded and used to calculate the relative standard deviation (RSD).

The accuracy of the method was evaluated by analyzing the recovery percentage of compounds in the extract of EAS. The recovery assays of the two flavonoids were carried out by adding known quantities of the standard solutions to known amounts of samples of EAS.

2.6 Sample analysis

Ten microlitres of each sample solution was injected into the instrument. Identification of the different compounds was made by comparing the HPLC retention times, UV absorptions and MS spectra with those of the standards. Quantification was performed on the basis of linear calibration plots of the UV absorption peak area at 360 nm against concentration.

3.1 Optimization of the HPLC–MS conditions

For LC–MS analysis, both positive and negative modes were attempted, only the negative mode afforded a high sensitivity of the target compounds.

3.2 Method validation

The results of the calibration curve and LOD were shown in Table 1. All the compounds displayed good linearity in the relatively wide concentration ranges.

The results of precision showed that RSDs of peak areas of rutin and quercetin were 1.629% and 0.493%, respectively. The RSDs of repeatability of the two compounds were 2.471% and 0.517%. In the stability test, the analytes were found to be rather stable within 24 h (RSD < 2.067%). The recoveries of rutin and quercetin were found in the range of 99.275–101.416% and 98.881–102.506%, and the RSDs of the recoveries were 0.905% and 1.362%, respectively.

3.3 Sample analysis

The spectroscopic and chromatographic characteristics of the compounds, including UV absorption, molecular ions, and LC retention times, were established under the experimental conditions for determining corresponding components in the samples.

The typical HPLC chromatograms of a mixed standard solution and sample solution collected from Heilongjiang Province were shown in Fig. 2. The UV and MS spectra of peaks 1 and 2 in the sample chromatogram were given in Figs. 3 and 4. The retention times, UVλ_max and MS signals of each compound were in good agreement to authentic compounds and reported literature values (Fang et al., 2008b; Lang et al., 2003; Wang et al., 2008).

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Figure 2

The HPLC chromatograms of the Heilongjiang sample (A) and the standards (B). Peaks: 1 = rutin; 2 = quercetin.

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Figure 3

The UV spectra of peaks 1 (A) and 2 (B).

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Figure 4

The MS spectra of peaks 1 (A) and 2 (B).

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Quantification was performed on the basis of linear calibration. The contents of the two flavonoids in different samples from ten origins were calculated and the results were shown in Table 2 with the mean values of three replicate injections. The results indicated that the contents in this herb from different locations were varied. Especially, the compound of rutin in Yunnan sample was not detected. The contents in different medicinal portions were also given in Table 2 and there were the fewest contents of the compounds in the root among other parts which meant that this part need not be used, so this herb can be better protected and utilized continually.