SIMULTANEOUS DETERMINATION OF SEVEN CONSTITUENTS IN HERBAL PRESCRIPTION JAEUMGANGHWA-TANG USING HPLC-PDA

A simple and accurate high-performance liquid chromatographic method was applied to the quantitative analysis of seven components of the traditional herbal prescription Jaeumganghwa-tang (JGT), including 5-hydroxymethyl-2furaldehyde, albiflorin, paeoniflorin, liquiritin, ferulic acid, nodakenin, and glycyrrhizin. All seven compounds were separated in less than 40 min on a Gemini C18 column at 40°C by gradient elution using 1.0% (v/v) aqueous acetic acid and acetonitrile containing 1.0% (v/v) acetic acid as mobile phase. The flow rate was 1.0 mL/min and the detector was a photodiode array (PDA) set at 230 nm, 254 nm, 280 nm, and 330 nm. The calibration curves showed good linearity (r > 0.9998) in different concentration ranges. The recovery of each component was in the range of 91.47–102.62%, with relative standard deviations (RSDs, %) less than 4.5%. The RSDs (%) for intraand interday precision were 0.06–2.85% and 0.06– 2.83%, respectively. The concentrations of the seven components in JGT were in the range 0.74–5.48 mg/g.


Chromatographic system
The HPLC system consisted of a Shimadzu LC-20A HPLC system (Shimadzu Co., Kyoto, Japan) with a solvent delivery unit, on-line degasser, column oven, autosampler, and PDA detector.The data processor used LCsolution software (Version 1.24, Shimadzu, Kyoto, Japan).The column used was a Gemini C18 analytical column (2504.6 mm; particle size 5 m; Phenomenex, Torrance, CA, USA).The mobile phases were solvent A (1.0% v/v aqueous acetic acid) and solvent B (acetonitrile with 1.0% v/v acetic acid).The gradient conditions are shown in Table 1.Column temperature was maintained at 40°C.Analysis was performed at a flow rate of 1.0 mL/min and monitored at 230 nm (for albiflorin and paeoniflorin), 254

Recovery
Recovery tests were performed by adding known amounts (low, medium, and high) of reference standards to JGT samples before extraction.An average recovery was calculated using the formula: Recovery (%) = (Amount determined -Amount original )/Amount spiked  100.

Precision and accuracy
Reproducibility was assessed by analysis of five independently prepared standard solutions.The relative standard deviation (RSD) of analyte peak areas and peak retention times for each standard were calculated.Intra-and inter-day precision values were determined using a standard addition method to prepare spiked samples, employing both standards and controls.

Optimization of chromatographic conditions
We obtained good separation chromatograms using mobile phases consisting of (A) 1.0% (v/v) aqueous acetic acid and (B) acetonitrile with 1.0% (v/v) acetic acid, with a gradient flow of 0-40 min, 5-70% B; 40-45 min, 70-100% B; 45-50 min, 100% B; 50-55 min, 100-5% B; and 55-70 min, 5% B. Quantitation was achieved by PDA detection in the region 190-400 nm, based on retention time and UV spectra compared with those of the standard.Using optimized chromatography conditions, all analytes eluted before 35 min, showed a resolution better than 1.75, and afforded good specificity upon sample analysis.Representative HPLC chromatograms of standards and the extract are shown in Figure 2.

Recovery
A recovery test was performed by addition of known amounts of 5-HMF, albiflorin, paeoniflorin, liquiritin, nodakenin, hesperidin, and glycyrrhizin to the extract.Standard compounds, at each of three different levels (low, medium, and high), were mixed with sample powder, and extracted.The recovery of each reference standard was in the range 91.47-102.62%,and the RSD range was 0.19-4.24%(Table 4).

Accuracy and precision
Reproducibility or intra-assay precision was assessed by repeatedly measuring retention times and peak areas for three independently prepared samples of analyte.Reproducibility for all analytes was less than RSD 1.0% for peak responses and less than RSD 0.1% for retention times (data not shown).Thus, the HPLC assay showed good repeatability under optimized conditions.To test the accuracy and precision of our analytical method, intra-and inter-day variations in measurement of seven major constituents were determined, and are summarized in Table 5.The intra-day accuracy was in the range 97.32-106.66%,and inter-day accuracy was 96.77-107.43%.b LOD = 3  signal-to-noise (S/N) ratio.c LOQ = 10  signal-to-noise (S/N) ratio.

Sample analysis
This newly established analytical method was applied to simultaneous determination of seven components in JGT.
In conclusion, we developed a simple and accurate HPLC method for simultaneous separation and determination of seven components, in order to evaluate the quality of JGT.This study is the first report of the simultaneous analysis of seven constituents in JGT using HPLC-PDA detection.In the present work, simultaneous determination of seven marker compounds in JGT was validated with respect to linearity, precision, and accuracy.The method will be helpful to improve the quality control and analysis of JGT.

Figure 1 :
Figure 1: Chemical structures of seven components of Jaeumganghwa-tang.

Table 1 :
Solvent gradient conditions for HPLC analysis.
and Zizyphi Fructus, were purchased from Omniherb (Yeongcheon, Korea) and HMAX (Jecheon, Korea).The origin of the samples was confirmed taxonomically by Prof. Je Hyun Lee and Young Bae Seo, Dongguk University, Gyeongju, Korea and Daejeon University, Daejeon, Korea, respectively.A voucher specimen (2008-KE01-1 through KE01-13) has been deposited at the Basic Herbal Medicine Research Group, Korea Institute of Oriental Medicine.
a Y: peak area (mAU) of components; x: concentration (g/mL) of components.

Table 4 .
Recovery levels of the seven marker compounds (n = 5).