Simultaneous Determination of Six Active Components in Danggui Kushen Pills via Quantitative Analysis of Multicomponents by Single Marker

In this paper, a valid evaluation method for the quality control of Danggui Kushen pills (DKP) has been established based on quantitative analysis of multicomponents by single marker (QAMS). Gallic acid, matrine, oxymatrine, catechin, ferulic acid, and rutin were selected as the indexes for quality evaluation of DKP. The analysis was achieved on an Agilent ZORBAX SB-C18 column (250  mm × 4.6  mm, 5 μm) via gradient elution. Gallic acid was used as internal standard to determine the relative correction factors (RCF) between gallic acid and other five constituents in DKP. The contents of those components were calculated at the same time. The accuracy of QAMS method was verified by comparing the contents of six components calculated by external standard (ES) method with those of the QAMS method. It turned out that there was no significant difference between the quantitative results of QAMS method and external standard method. The proposed QAMS method was proved to be accurate and feasible according to methodological experiments, which provided an accurate, efficient, and economical approach for quality evaluation of DKP.


Introduction
Danggui Kushen pills are a classical compound consisting of Angelica sinensis and Sophora flavescens, which have the effect of cooling blood and expelling dampness. It can be used for treatment of head and face sores, acne and pimples, eczema and itching, wine trough nose caused by blood dryness, and dampness heat [1][2][3]. In the "Ministry-Issued Traditional Chinese Materia Medica Preparation," thin-layer chromatography (TLC) method was applied to identify the two herbs, Angelica sinensis and Sophora flavescens, in DKP. It was found that HPLC method was mainly used to control the quality of DKP through consulting literature. In addition, only ferulic acid, the active ingredient in Angelica sinensis, or matrine and oxymatrine, the main active component in Sophora flavescens, were determined [4][5][6]. However, other active ingredients with anti-inflammatory and antibacterial effects in this preparation, such as gallic acid, catechin, and rutin, are of equal importance in the treatment of acne and pimples [7][8][9]. erefore, relying on single or several components to assess the quality of DKP is insufficient. It is extremely necessary to establish a low-cost, reliable, and efficient approach for quality evaluation of DKP. QAMS method has been considered as a good alternative for the quality control of traditional Chinese medicine and its preparation [10][11][12]. In this paper, a QAMS method for simultaneous determination of gallic acid, matrine, oxymatrine, catechin, ferulic acid, and rutin in DKP was established. Gallic acid was used as the internal reference standard due to its low price and easy acquisition. e proposed QAMS method laid foundation for the establishment of quality standard of DKP as well. e chemical structure of those six active components is shown in Figure 1.

Experimental
2.1. Instrument. Chromatographic separation was achieved on an Agilent 1260 high performance liquid chromatography (HPLC) system (including quaternary low pressure mixing pump, autosampler, column oven, diode array detector, and Chemstation workstation). An AB135-S electronic balance was obtained from Mettler Toledo International Co., Ltd. A DK-98-II type electric-heated thermostatic water bath pot was purchased from Tianjin Taisite Instrument Co., Ltd. A KQ-250 ultrasonic cleaner was purchased from Kunshan Ultrasonic Instrument Co., Ltd. An R series rotatory evaporator was acquired from Shanghai Shen Sheng Technology Co., Ltd.  e solution was shaken well and filtered through a 0.22 μm membrane filter before HPLC analysis.

Preparation of Test Solution.
About 5 g DKP was grinded and placed in a stoppered conical flask. en 100 mL methanol was added into the conical flask and was ultrasonically extracted for 30 min. Ultrasonic extraction was repeated for three times. e extracts of each time were filtered and combined together. e combined filtrates were concentrated and evaporated to dryness via roller evaporator. Finally, the residue was redissolved with methanol (final adjusted volume 10 mL). e solution above was filtered through a 0.22 μm microporous membrane filter after full shaking, and subsequent filtrate was collected as the test solution.

Preparation of Negative Control Solution.
According to the prescription proportion, negative control samples without Sophora flavescens or Angelica sinensis-Sophora flavescens were taken and prepared into negative control solutions, respectively, based on the method in Section 2.4.

Chromatographic Conditions.
Analysis was achieved on the Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm) along with the detective wavelength of 210 nm, 225 nm, 256 nm, and 320 nm. e mobile phase consisted of methanol (A) and 0.1% phosphoric acid solution (B). e flow rate was 1.0 mL·min −1 , and the column temperature was maintained at 30°C. e injection volume of sample was 5 μL. e concrete gradient elution conditions are displayed in Table 1

Linear Range.
e stock standard solution (precise 0.1 mL, 0.25 mL, 0.5 mL, 1 mL, 2 mL, and 3 mL) was placed in a 5 ml volumetric flask and was dissolved with methanol (final adjusted volume 5 mL), respectively, to obtain the standard serial working solutions. en the above working solutions were injected into the HPLC for analysis according to the chromatographic conditions under Section 2.6, respectively. In addition, the mixed standard solution was diluted with methanol. e limits of detection (LOD) and the limits of quantification (LOQ) were determined by three times and ten times of the signal noise ratio, respectively.

Stability
Test. An aliquot of same test solution (placed at room temperature) was injected into HPLC at 0, 2, 4, 8, 12, and 24 h for analysis according to the chromatographic conditions under Section 2.6, respectively. e peak area of each component was recorded, and RSD was calculated.

Repeatability Test.
Six aliquots of DKP with the same batch number were taken and prepared into six parallel test solutions following the method under Section 2.4 and injected into HPLC for analysis according to the chromatographic conditions under Section 2.6.

Recovery Test.
Six copies of 2.5 gram DKP(batch number: 20171201) with known content were accurately weighed and placed in stoppered conical flasks, respectively. en 1 mL mixed standard solution (containing matrine 3120 μg, gallic acid 30064 μg, oxymatrine 7.5 μg, catechins 105.4 μg, rutin 40.3 μg, and ferulic acid 1322.7 μg per mL) was added into the conical flasks above and prepared respectively. e sample solutions were prepared according to the method under Section 2.4 and injected into HPLC for analysis following chromatographic conditions under Section 2.6, respectively. e peak areas were recorded, and average recovery rate and RSDs of each sample were calculated.

Specialization Test.
e theoretical plates were selected according to the separation resolution between the analytes and impurities. After several tests, we found that the analytes have good resolution in chromatographic peaks when theoretical plates are bigger than or equal to 3000. As shown in Figures 2-5, there was no interference in the corresponding position of the six components. In addition, the theoretical plate numbers of those constituents were not less than 3000. e separation degrees were all greater than 1.5, which indicated that the proposed method was of good specialization.

Linear Range.
e standard curve was drawn by using the chromatographic peak area (Y) as the vertical axis and the concentration of the reference solution (X) as abscissa. In addition, the limit of detection (S/N � 3) and the limit of quantification (S/N � 10) were calculated. e results are shown in Table 2, suggesting that six components presented good linear relationships in their determination ranges. LOD and LOQ of six substances were within the range of 0.01-0.11 μg·mL −1 and 0.04-0.31 μg·mL −1 , which showed a high sensitivity under the established chromatographic condition.

Calculation of RCF.
e RCF was calculated by multipoint correction method using the following calculation formula: where f s/k is the RCF of component to be measured, A s is the peak area of internal reference, W s is the concentration of internal reference, A k is the peak area of component to be measured, and W k is the concentration of component to be measured. Gallic acid was selected as the internal reference (1.000) for the quantitative analysis of other five components (matrine, oxymatrine, catechin, ferulic acid, and rutin) under the detection wavelengths of 210 nm, 225 nm, 256 nm, and 320 nm, respectively. e results are displayed in Table 3. It was found that the RCFs of matrine, oxymatrine, and catechin were 3.80, 3.03, and 0.39 under 210 nm. e RCF of rutin was 2.31 under 256 nm. e RCF of ferulic acid was 2.38 under 320 nm.

Reproducibility Test of RCF.
e results showed that RSD values of RCF measured in different conditions were all less than 2.27%, which indicated that the RCF calculated by the proposed method was of good reproducibility.

Location of the Chromatographic Peak of Measured
Component. e relative retention time was calculated according to the ratio of retention time of measured component (k) and internal reference (s). e calculation formula (2) is shown as follows: where Rt R is relative retention time, t Rk is the retention time of component to be measured, and t Rs is the retention time of internal reference. Rt R between measured components and internal reference (gallic acid) was calculated via formula (2). In addition, RSDs of relative retention time measured in different conditions were calculated in order to evaluate the influence of different chromatographic systems, chromatographic column, column temperatures, and flow rates on Rt R . e results showed that RSDs of relative retention time between tested components and internal reference were all less than 1.65%, indicating that Rt R was of good reproducibility and can be used to locate of the chromatographic peak of other five analytes. On the other hand, overall shape of chromatographic spectra of measured components can also be applied to the location of chromatographic peaks. In a word, chromatographic peaks can be accurately located in a great extent by the method established above.

Comparison of the Results of QAMS Method and ES
Method. e contents of each component in five batches of DKP were calculated by ES and QAMS method, respectively. e results are exhibited in Table 4. It was found that the relative errors (RE) of these two methods were less than 3.0%.
ere were no significant differences between the results of two determination methods, illustrating that the method established above was accurate and reliable.
3.3. Discussion. Traditional Chinese medicine compound preparations, as a traditional and classic medicine, have the characteristics of the diversity and complexity of traditional Chinese medicines as well. erefore, the method for determining the content of traditional Chinese medicine compound preparations is gradually improving. e QAMS method has been widely used in traditional Chinese medicines and their decoction pieces and preparations [13][14][15]. Simultaneous determination of multiple components can be achieved via measurement of single component utilizing the function or proportion relationship between various components of traditional Chinese medicines. In this paper, QAMS method was applied to establish a new method for simultaneous determination of matrine, gallic acid, oxymatrine, catechin, ferulic acid, and rutin in DKP, which provided a better and convenient way to control the quality of DKP.
Gallic acid was selected as the internal reference of QAMS method in this study because gallic acid is one of the active ingredients of DKP and has pharmacological effects such as anti-inflammatory, antibacterial, antipathogenic microorganisms, antivirus, and so on. Gallic acid also has the advantage of low price and easy acquisition. Furthermore, there were no significant differences between the content determination results of QAMS method and ES method when gallic acid was used as internal reference. Taking the retention time and content of other components to be tested into consideration, gallic acid was considered as the internal reference finally. e retention time of other measured components cannot be confirmed when QAMS method was applied because only one standard substance, internal reference (gallic acid), was used. erefore, it was critical to locate the chromatographic peaks of components to be tested. In this study, retention time difference method and relative retention value method were investigated [16,17]. e results showed that RSDs of retention time difference and relative retention value determined in different conditions were less than 6.06% and 1.65%, respectively. So, relative retention value method was used to locate the chromatographic peaks of components to be tested due to its good reproducibility and feasibility.

Conclusion
In this paper, the HPLC-DAD method was adopted for determination of relative retention time and relative correction factor of six active components in DKP for the first time. A QAMS method was developed for simultaneous determination of six active components in DKP. e QAMS method proposed in this study has been proved by methodology validation and found to be accurate, feasible, and reliable for contents determination of six active components in DKP, which can be applied to the quality control of DKP.

Data Availability
e data used to support the findings of this study are included within the Supplementary Materials.    instruments, chromatographic column, column temperature, and flow rate. Table 5: the effects of different instruments and chromatographic column on relative retention time (tR) and retention time difference (Δt) of six tested components. (Supplementary Materials)