Development and Validation of an HPLC Method for Simultaneous Determination of Nine Active Components in ‘ Da-Chai-Hu-Tang ’

In this study, a simple, reliable and accurate method for the simultaneous separation and determination of naringin, hesperidin, neohesperidin, baicalin, wogonoside, baicalein, wogonin, emodin and chrysophanol in ‘Da-Chai-Hu-Tang’ was developed by reverse-phase high-performance liquid chromatography (RP-HPLC). The chromatographic separation was performed on an Agilent ZORBAX C18 column (250 mm × 4.6 mm i.d., 5.0 μm), and the mobile phase composed of methanol and water containing 1% (v/v) acetic acid was used to elute the targets in a gradient elution mode. The flow rate and detection wavelength were set at 0.8 ml/min and 280 nm, respectively. All calibration curves of the nine components expressed good linearities (r2 ≥ 0.9992) within the tested ranges. The RSD values demonstrated the intraand inter-day precisions were less than 2.89%, and the recoveries of the investigated compounds were between 96.22% and 105.28%. The proposed method is simple, precise, specific, sensitive, and successfully applied to determine the nine marker compounds in ‘Da-Chai-Hu-Tang’ for quality control.


Introduction
Traditional Chinese medicines (TCMs), especially in China, have played an important role in clinical therapy, and been widely used for the prevention and treatment various diseases because of its high effectiveness and low toxicity for thousands of years [1,2].
Generally, herbal medicines are used in combinations to afford a formula composed of several single herbs.Combining the herbs together and boiled in solvent can make different preparations, and multiple constituents are usually responsible for the therapeutic effects by synergistic or antagonistic interactions.Each herb has its own bioactivities, but when many herbs are combined, there may be changes of active components, especially in their contents.Moreover, some TCMs have been widely administrated directly after boiling with water without any quality assessment in some areas of China, which may produce some side effects and influence the activities of herbal products because of different herbs from different regions with different contents of active com-pounds.That is why the quality of TCMs is very critical important for affording the efficiency and avoiding the toxicity.Thus, sensitive and reliable holistic analytical approach is necessary.
Mostly, single marker compound is analyzed to evaluate the quality of TCMs [3], which is simple but cannot totally demonstrate the quality of herbal prescriptions.Then, multiple components analysis (MCA) method has been developed, which can simultaneously evaluate many active compounds from different herbs and has been widely used for the quality control of TCMs [4][5][6].
In the process of component determination, analytical methods and technologies are essential.Up to date, two kinds of chromatographic techniques, high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry (HPLC-MS), have been used more and more frequently for the quality control of various kinds of herbal medicines [7][8][9].The former, has been universally used as a convenient and sensitive method because of its convenience, precision, cheapness, sensitivity and reproducibility [10].The later, can screen the chemical constitu-ents high-throughput in TCMs, especially those trace components which are difficult for analysis by conventional methods.Hence, HPLC-MS is a powerful tool for its high level of sensitivity and selectivity, but the expensive running cost violates its application in routine analysis.Thus, in this paper, HPLC method was established to achieve quality control.
The Chinese formular 'Da-Chai-Hu-Tang' (DCHT), is a botanical drug and composed of Radix bupleuri, Fructus aurantii immaturus, Rhizoma zingiberis recens, Radix scutellariae, Radix paeoniae alba, Rhizoma pinelli and Fructus jujubae.Because of its therapeutic effectiveness and few side effects, DCHT has been widely used to treat acute cholecystitis, cholelithiasis, pancreatitis and appendicitis in China [11].By now, pharmacological research has demonstrated that it also shows a good effect in inhibition atherosclerosis and fatty liver [12,13].
The aim of the present paper was to establish a simple, efficient and sensitive method for simultaneous analysis of nine marker compounds including naringin, hesperidin, neohesperidin, baicalin, wogonoside, baicalein, wogonin, emodin and chrysophanol (shown in Figure 1) in DHCT for quality control by HPLC.

Materials and Reagents
Nine standard compounds of naringin, hesperidin, neohesperidin, baicalin, wogonoside, baicalein, wogonin, emodin and chrysophanol were purchased from the National Institute for Control of Pharmaceuticals and Biological Products (Beijing, China).Medicinal plants, Radix bupleuri, Fructus aurantii immaturus, Rhizoma zingiberis recens, Radix scutellariae, Radix paeoniae alba, Rhizoma pinelli, Fructus jujubae and Radix et Rhizoma Rhei were purchased from a local drug store (Dalian, China) and identified by Dr. Yun-Peng Diao (Dalian Medical University, Dalian, China).Voucher specimens were deposited in College of Pharmacy, Dalian Medical University (Dalian, China).Methanol was HPLC grade (TEDIA, USA), and water for HPLC analysis was prepared using a Millipore (Millipore, USA).Acetic acid and other reagents were analytical grade purchased from ShenLian Chemical Factory (Shenyang, China).All the solvents and solutions were filtered through a Millipore filter (0.45 μm) before use.

Standard Solution Preparation
A mixed stock standard solution containing naringin, hesperidin, neohesperidin, baicalin, wogonoside, baicalein, wogonin, emodin and chrysophanol was prepared by accurately weighing appropriate amounts of the nine reference compounds and dissolving in methanol.All the standard stock and working solutions were prepared in dark brown calibrated flasks and stored at 4˚C.

Preparation of Sample Solutions and Negative Control Solutions
Ten medical plants were triturated into powders in the particle size of 40-60 mesh, and then weighed according to DCHT formula and blended.The mixed powder (0.70 g) was extracted by 20 ml methanol for 20 min in an ultrasonic bath.In order to keep the repeatability of the extraction procedure, lost volume of methanol was compensated after extraction.After filtration, 2 ml filtrate was transferred into a 10 ml volumetric flask with MeOH and 10 μl of the resultant solution was injected into the LC system for analysis after through a 0.45 μm Millipore filter.
According to the prescription and preparation protocol of DCHT formula, three kinds of negative control samples in which the formula without F. aurantii Immaturus, R. scutellariae, or R. et Rhizoma Rhei, respectively, were prepared to validate the specificity of the method.The negative samples were prepared according to the method mentioned above.

Apparatus and Chromatographic Conditions
Chromatography was performed with an Agilent Technologies 1200 series HPLC system consisting of a quaternary delivery system, an auto-sampler and a DAD detector.All the separations were carried out on a ZORBAX SB C18 column (250 mm × 4.6 mm I.D., 5 μm).The mobile phase consisted of methanol (A) and water containing 1% acetic acid (B) at a flow rate of 0.8 ml/min with a gradient elution mode was carried out as follows: 0-20 min, linear gradient from 15% A to 35% A; 20-40 min, the mobile phase was held on 35% A; 40-60 min, linear gradient to 50% A; 60-110 min, the linear gradient to 80% A; 110-120 min, the linear gradient to 95% A. Each run was followed by equilibration time of 15 min.Ultraviolet (UV) spectra were monitored at 280 nm.All the data were collected and analyzed with Chemstation software.

Optimization of Chromatographic Conditions
To develop an accurate, valid and optimal chromatogram, some HPLC analytical parameters including separation column, mobile phase and its elution mode were all investigated in this study.

Optimization Sample Extraction Protocol
The

Specificity of the Method
In order to investigate the specificity of the method, different negative control samples of DHCT were prepared and analyzed by HPLC, and the chromatograms are shown in Figure 4.It was obvious that there were no interferences for determination of the nine compounds by comparing the retention times with the standards.Furthermore, the purities of the investigated peaks were all confirmed to be pure through DAD purity studies.

Calibration Curves, the Limit of Detection (LOD) and Quantification (LOQ)
The external standard method was used to obtain regression  equations.The calculated results are shown in Table 2.
In the regression equation y = ax + b, y refers to the peak area, x is the concentration of the standard compounds (µg/ml), while a is the slope rate of the line and b is the intercept of the straight line with y-axis.All the standard compounds showed good linearity (r2 ≥ 0.9992) in the tested concentration ranges.The limit of detection (LOD) and quantification (LOQ) were also measured.The standard solution was diluted with MEOH to the appropriate concentrations.The detection limit was defined as the lowest concentration level resulting in a peak area of three times the baseline noise.LOD was in the range of 0.07-0.30µg/ml.The LOQ was obtained as amount to give a signal-to-noise ratio (S/N) of 10 in the range of 0.35-0.87µg/ml (listed in Table 2).a y is the peak area in HPLC analysis monitored at 280 nm, x is the concentration of compound (μg/ml); b LOD refers to the limit of detection, S/N＝3; c LOQ refers to the limit of quantification, S/N＝10.

Assay Precision, Repeatability, Stability and Recovery
The precision of the method was validated by both intraand inter-day precisions.The assays were carried out on the same mixed standard solutions at low, medium and high concentration levels during one day and one assay each day for three consecutive days, respectively.Relative standard deviation (RSD) of the mean content for each compound was calculated and ranged from 0.46% to 2.89% for intra-and inter-day precisions, which is shown in Table 3.The results indicated that the accuracy and precision of the proposed method were sufficient for determination of the nine compounds in the sample of DCHT.
The analysis repeatability of the nine components (Table 4) was determined by analysis of six samples which were prepared with the same preparation procedure and processed in parallel as described above.The RSD was calculated as a measurement for the repeatability of the method.The results indicated that the RSD values of each compound detected were all less than 1.97%, which showed good reproducibility of the developed method.
For the stability test, a sample of DCHT was analyzed with the interval of 6 h (0, 6, 12 and 18 h) at room temperature, and the sample solution was found to be stable (RSD values of the mean content were lower than 2.15%).The results are listed in Table 4.
The recovery assays were carried out by adding known contents of the standard samples to the known amounts of samples of DCHT and comparing the determined amount of these standards with the amount originally added.Table 4 shows these results of recovery tests.The mean recovery of the method was in the range of 96.22-105.28%,with RSD of less than 2.33%.Considering the results of the recovery assays, the method was thus acceptable.

Conclusions
An HPLC method for simultaneous determination of nine active compounds including rhaponticin, naringin, hesperidin, neohesperidin, baicalin, wogonoside, baicalein, wogonin, emodin and chrysophanol in DHCT has not been reported.The presented method in addition to its novelty for determination of nine ingredients was sufficiently rapid, simple and sensitive as well as precise and accurate, and it was not interfered with other chemical constituents in DCHT.The linearity, accuracy, precision, LOD and LOQ, specificity-selectivity of the method and sample stability were all established.Although nine compounds were quantitated, there are many other components in DCHT.More researches can be practiced for further investigation.But the method has several advantages, including rapid analysis, simple mobile phase, and simple sample preparation.It was success-fully used for the analysis of compatibility study of a formulation prepared in our laboratory and suitable for routine analysis in quality-control laboratories.

Acknowledgements
This research was partially supported by the excellent young scientists funds (No.2006 J23JH024) of the Science and Technology Foundation of Dalian, China.

Figure 3 .
Figure 3. Efficiencies of the extraction for the nine compounds in DCHT using different: (a) extraction solvent; (b) the use of methanol; (c) extraction time.

Figure 4 .
Figure 4. Representative HPLC chromatograms of: (a) the negative sample without F. aurantii immaturus; (b) the negative sample without R. scutellariae; (c) the negative sample without R.et Rhizoma Rhei.

Table 1 . The tried column and mobile phase in optimization of HPLC conditions.
extraction conditions, for example extraction solvent, method and time, can easily influence the efficiency of the extraction.As a result, it is necessary to estimate and optimize the factors affecting extraction recovery.Two methods, boiling and ultrasonic are often used to extract the targets from matrix.The disadvantages of the boiling procedure are the loss of the compounds due to ionization, hydrolysis and oxidation during extraction, the consumption of a large amount of solvent, low extraction efficiency, and time-consuming.These shortcomings have led to the consideration of ultrasound-assisted extraction (UAE) method, which has been widely used in quality control of TCMs.In UAE process, extraction solvent, sample-solvent ratio and extraction time are critical important for high extraction efficiency.Methanol is often used as the extraction solvent because of its high efficiency and directly application for