Set up of the RP-HPLC-IPAD system
The RP-HPLC-IPAD system had the following composition for analysis of furanocoumarins in Radix Angelicae Dahuricae. The mobile phase, composed of 50% ACN and water, flowed according to the gradient elution program on a C-18 column with a 0.11 mL/min flow rate. The eluent was mixed with NaOH solution before entering the detector. A post-column eluent was used to supply NaOH to the mixer. The post-column eluent concentration and flow rate were set to 100.0 mM NaOH and 0.80 mL/min, respectively. This system delivered a high-sensitivity analysis of furanocoumarins.
The sensitivities of six furanocoumarins were investigated using Hypersil Gold C-18 (25 × 0.46 cm I.D.; 5.0 μm) (4.6 mm column) and Cadenza C-18 (25 × 0.15 cm I.D.; 3.0 μm) (1.5 mm column) column types (Fig. 1(A)). The flow rates for the two columns were 1.0 and 0.11 mL/min, respectively. The IPAD method was used to measure the current generated by oxidation of a component at more than one potential at a gold electrode in a strongly basic medium (pH > 11). The IPAD sensitivity decreased as ACN volume increased because ACN suppressed detection (Kwon et al. 2008). Our RP-HPLC-IPAD system is a post-column delivery system in which the mobile phase and NaOH are mixed in a mixer. A column of large diameter supplies a large volume of ACN; therefore, IPAD sensitivity decreases with increase in column diameter at a set post-column NaOH flow rate (Kwon et al. 2008). Figure 1(A) shows that the 4.6 mm column had lower baseline stability and sensitivity than the other column. The sharp target peaks of the chromatogram were the basis for selection of the 1.5 mm column for further analyses.
The sensitivities of six furanocoumarins were investigated according to waveforms (triple-, quadruple- or six-potential) (Fig. 1(B)). All target peaks on the chromatogram were adjusted based on an internal standard peak. The peaks with the quadruple-potential waveform had 1.4–2.4-fold better sensitivity than those with the triple-potential waveform. Analysis of glycosides or amino acids usually uses the six-potential waveform of E1 = -0.200 V, E2 = 0.000 V, E3 = +0.220 V, E4 = 0.000 V, E5 = -2.000 V, and E6 = +0.600 V. While glycosides with sugar moieties measured through E3 = +0.220 V were highly sensitive, furanocoumarins without a sugar moiety were not. Thus, it was necessary to increase the potential of E3. Furanocoumarins were detected at high sensitivity by increasing the potential of E3 to 0.550 V. The peaks with our six-potential waveform had 2.0–8.2-fold better sensitivity than those with the triple-potential waveform. Our IPAD method enabled the first high-sensitivity analysis of furanocoumarins.
Optimization of extraction efficiency
Water, 50% EtOH, 50% MeOH, and 50% ACN were tested as extraction solvents for furanocoumarins in Radix Angelicae Dahuricae, as shown in Figure 2(A). Among them, 50% EtOH with the highest extraction efficiency was suitable as the solvent. Figure 2(B) shows the extraction efficiencies according to extraction method (sonication or reflux). The 50% EtOH was used as an extraction solvent, and the Radix Angelicae Dahuricae was extracted under sonication or reflux for 60 min. Oxypeucedanin hydrate and byakangelicin were soluble in water, while the others were soluble in 50% EtOH. The extraction efficiencies of the sonication method were lower than those of the reflux method for oxypeucedanin hydrate and byakangelicin; the same for bergapten; and higher for byakangelicol, imperatorin, and isoimperatorin. For optimal total extraction amount, the sonication method was higher than the reflux method and was selected. Figure 2(C) shows the extraction efficiencies according to extraction time. The Radix Angelicae Dahuricae was extracted with 50% EtOH through the sonication method for 30, 60, 90, or 120 min. The extraction efficiency was highest with the 60 min extraction. Based on these results, Radix Angelicae Dahuricae was set to be extracted for 60 min with 50% EtOH using the sonication method.
Method validation
Sensitivity and linearity
The linearities of the six target components were confirmed using four standard calibration points (LOQs [oxypeucedanin hydrate, 0.1 ng; byakangelicin, 0.05 ng; bergapten, 1.0 ng; byakangelicol 0.1 ng; imperatorin, 0.005 ng; isoimperatorin, 1.0 ng], 20.0 ng, 100.0 ng, and 500.0 ng). Linear equations and ranges are shown in Table 1. The coefficients of determination were 0.9995 - 1.0000. The IPAD method has 1.05–110-fold better sensitivity than the UV method (Table 1). Figure 3 shows comparative chromatograms of the UV and IPAD methods for Radix Angelicae Dahuricae extracts. Our IPAD method produced much better stability and noise reduction of the baseline than did the UV method. While the UV method sensitivity was too low to analyze imperatorin and isoimperatorin, our IPAD method had sufficient sensitivity. Our IPAD chromatogram showed mostly sharp peaks, and byakangelicol and its neighboring unknown peak had good separation. However, the UV chromatogram showed broad peaks, with that of byakangelicol and its neighboring unknown peak overlapping. Therefore, our IPAD method had better sensitivity, peak sharpness, and baseline stability compared to the UV method for analyzing Radix Angelicae Dahuricae.
Accuracy and precision
The inter- and intra-day tests were performed by measuring samples daily for five consecutive days (Table 2). The relative standard deviations (RSDs) were 0.1%–4.2% in the inter-day assay and 0.8%–4.9% in the intra-day assay. The recovery test was conducted for accuracy evaluation of our method. The RSD ranges and mean recoveries were 0.5%–4.8% and 96.4%–104.5% for Radix Angelicae Dahuricae (Table 3), showing that this method has excellent precision and accuracy.
Determination of six furanocoumarins in Radix Angelicae Dahuricae and GMGHT
We quantified six furanocoumarins in Radix Angelicae Dahuricae and GMGHT through our IPAD method. The samples were used in powdered form. Figure 4 shows the extraction efficiency. Radix Angelicae Dahuricae was extracted under reflux for 1 h with 50% EtOH or water and analyzed. Oxypeucedanin hydrate and byakangelicin were soluble in water, and the boiling point in water was higher than that in 50% EtOH. The extraction efficiencies for oxypeucedanin hydrate and byakangelicin in water were 4.0- and 3.6-fold higher, respectively, than those in 50% EtOH owing to the solubility differences. In contrast, bergapten, imperatorin, and isoimperatorin were sparingly soluble in water and soluble in 50% EtOH. The extraction efficiencies for bergapten, imperatorin, and isoimperatorin in 50% EtOH were 3.8-, 21.8-, and 66.4-fold higher, respectively, than those in water. The 50% EtOH had the best total extraction efficiency. However, because GMGHT usually is taken in liquid form by mouth, water instead of 50% EtOH was judged to be more suitable as an extraction solvent for Radix Angelicae Dahuricae or GMGHT.
Table 4 shows the results of quantification of the six furanocoumarins in Radix Angelicae Dahuricae and GMGHT. In general, the extraction efficiency of target components from a mix of several medicinal herbs, such as that in GMGHT, is lower than that from a single medicinal herb, such as Radix Angelicae Dahuricae. In the former case, we used the correction factor for transfer (CFT) (maximum amount of a target component that can be extracted from the Chinese medicinal preparation / measured amount of the target component extracted from the Chinese medicinal preparation). The CFTs for oxypeucedanin hydrate, byakangelicin, and byakangelicol were in the range of 3.13–11.40. The extraction efficiencies of oxypeucedanin hydrate, byakangelicin, and byakangelicol from GMGHT were lower than those from Radix Angelicae Dahuricae. It was inferred that the presence of the other medicinal herbs in GMGHT lowered the extraction efficiency. In contrast, the CFTs for bergapten, imperatorin, and isoimperatorin were in the range of 0.07–0.86, beyond the maximum values expected. Notopterygii Rhizoma (Kim et al. 1989) and Ledebouriellae Radix (Kim et al. 2002) have been reported to contain bergapten and imperatorin, and Notopterygii Rhizoma has been reported to contain isoimperatorin (Kim et al. 1989). Notopterygii Rhizoma and Ledebouriellae Radix are medicinal herbs included in GMGHT. The CFT values of these were low because bergapten, imperatorin, and isoimperatorin were present in large quantities in Rhizoma Notopterygii and Radix Ledebouriellae. Based on these results, oxypeucedanin hydrate is the most useful marker of Radix Angelica Dahuricae in GMGHT, for which our method is suitable for quality control.
Figure 5 shows the IPAD chromatograms of GMGHT and Radix Angelica Dahuricae. Both show good separation of all target components without overlap of unknown peaks. Quantification of the six furanocoumarins from Radix Angelica Dahuricae or GMGHT was possible without a complex pretreatment.