Simultaneous Determination of Eight Chemical Components in Angelicae Sinensis Radix and Its Herbal Products by QAMS

A HPLC method has been developed for simultaneously detecting chlorogenic acid, ferulic acid, senkyunolide I, senkyunolide H, coniferyl ferulate, senkyunolide A, ligustilide, and levistolide A in Angelicae Sinensis Radix through quantitative analysis of multicomponents by single-marker (QAMS) method with ferulic acid as internal standard substance. The relative analysis correction factors of each component in Angelicae Sinensis Radix have good reproducibility under different chromatography conditions. In addition, no significant difference of results was found between quantitative analysis of multicomponents by single-marker (QAMS) method and external standard method in determining content of these components of different Angelicae Sinensis Radix and its 12 kinds of preparations. As a result, the established QAMS method for Angelicae Sinensis Radix analysis with ferulic acid as internal standard substance is accurate and feasible, which could be used as an effective and economical method to control quality of Angelicae Sinensis Radix and its herbal products.


Introduction
Angelicae Sinensis Radix (ASR, named Danggui in Chinese) is the dried root of Angelica sinensis (Oliv.) Diels [1]. As a Chinese herbal medicine, ASR was originally described in an ancient traditional Chinese medicine classic named Shennong's Classic of Herbology, in which it is classified as top grade [2][3][4]. It is one of the most common traditional Chinese medicines (TCM) in our country and used in more than 80 composite formulae. ASR has the functions of tonifying blood circulation, activating blood circulation, regulating menstruation, relieving pain, moistening intestines, and defecating. It is widely applied for blood deficiency, vertigo palpitations, irregular menstruation, amenorrhea and dysmenorrhea, deficiency and cold abdominal pain, rheumatic arthralgia, intestinal dryness constipation, and other diseases [5][6][7]. ASR is widely distributed in Gansu, Sichuan, Hubei, Shanxi, and other provinces of China. It is not only exploited as a healthy food and drug in Asian countries but also as a nutrient in women's health and known as "female ginseng" in Europe. e preparations of ASR are mainly produced as Danggui pills, Danggui Kushen pills, Tiaojing Zhitong tablets, Fuke Tiaojing tablets, and so on [8][9][10].
ASR mainly contains volatile oil, organic acid, polysaccharide, brain glycosides, nucleosides, amino acids, and other types of compounds. Furthermore, over 70 compounds have been identified, including essential oils (such as ligustilide, butylphthalide and senkyunolide, and phthalide dimers), organic acids and their esters (such as ferulic acid and conifer ferulate), and vitamins and amino acids [11][12][13][14]. Among these compounds, ferulic acid has varieties of bioactivities, which has been utilized as the marker compound for quality evaluation of ASR in Chinese Pharmacopoeia. However, the characteristics of TCM determination are "multicomponents, multisites, multiefficacy, and multitargets," so it is difficult to express and evaluate the quality of TCM scientifically, effectively, and comprehensively based on a single index component [15][16][17][18]. So, the multicomponent analysis is necessary. But these methods have some difficulties in the practical application [19][20][21] such as the requirement of expensive standard substances, the difficulty to achieve the separation of the components, or the instability of the monomer, among others. e quantitative analysis of multicomponents by single-marker (QAMS) method is an analytical method for multi-index quality evaluation model suitable for the characteristics of TCM. e method used a cheap and easily available component as an internal standard substance to establish the relative calibration factors (RCF) between the internal standard substance and other components that are to be tested so that the simultaneous determination of multiple components to be tested can be realized. To some extent, this method could also reduce the analysis cost of quality control of TCM [22][23][24].
As known to us, the simultaneous determination of multicomponents in ASR is mainly concentrated on ferulic acid, ligustilide, and senkyunolide I through using high performance liquid chromatography (HPLC). It could not make a comprehensive and scientific evaluation of ASR and its herbal product [25,26]. erefore, the QAMS was used to simultaneously determine ferulic acid, chlorogenic acid, senkyunolide I, senkyunolide H, coniferyl ferulate, senkyunolide A, ligustilide, and levistolide A in this study, the ferulic acid was used as internal standard substance, and the RCF of other compounds was established. e established method could simultaneously determine the contents of 8 active components (Figure 1) in ASR and its 12 kinds of preparations, which provides a theoretical scientific basis for the total quality control and evaluation of ASR and its preparations.

Preparation of Mixed Standard Solution.
Chlorogenic acid, ferulic acid, senkyunolide I, senkyunolide H, coniferyl ferulate, senkyunolide A, ligustilide, and levistolide A were accurately weighed 4.7 mg, 11.1 mg, 2.9 mg, 3.7 mg, 5.4 mg, 9.7 mg, 22.2 mg, and 5.8 mg and poured into a 5 mL volumetric flask to make the concentration to 0.94, 2.22, 0.58, 0.74, 1.08, 1.94, 4.44, and 1.16 mg/mL of reference stock solution, respectively. Each reference stock solution was suck accurately and put into a 5 mL measuring flask, and then methanol was added to the scale and we shook them up; the concentration to 0.0564, 0.0444, 0.0116, 0.0074, 0.0648, 0.0155, 0.7104, and 0.0116 mg/mL of the mixed reference solution was finally obtained.

Preparation of Test Solution.
Dried plant material (over 40 mesh sieve, 2.00 g) was extracted exhaustively with ethanol/water (80/20, v/v) (50 mL) by an ultrasonic extractor (30 min, 40°C); we waited for the extracted samples to cool and then weighted and ethanol/water (80/20, v/v) was added to make the weight of samples reach as high as the weight of samples before extracting, shook it well, and filtered through 0.22 μm Millipore membranes.

Chromatographic Conditions.
e mobile phase was acetonitrile (A)-1% glacial acetic acid solution (B), gradient elution (0-18 min, 0%-19% A; 18-60 min, 19%-100% A); the flow rate was 1.0 mL/min, the column temperature was 30°C, the detection wavelength was 280 nm, and the sample volume was 10 μL. Under the conditions mentioned above, the chromatographic peaks of chlorogenic acid, ferulic acid, senkyunolide A, senkyunolide H, senkyunolide I, coniferyl ferulate, ligustilide, and levistolide A were separated well. e HPLC chromatogram of the mixed reference and Angelica test solution is shown in Figure 2

Method Validation
3.1.1. Linear Range. 1, 3, 7, 10, 13, and 18 μL of the mixed control solutions were taken to determine the linear correlation under the abovementioned chromatographic conditions. e results suggested that 8 components presented good linear relationships in their determination ranges (Table 1).

Precision.
e mixed control solution under "Section 2.3" was injected in 10 μL for 6 times continuously, the peak area of each component was recorded, and the relative standard deviation (RSD) of each component was 0.38%, 0.37%, 0.94%, 0.57%, 0.16%, 0.20%, 0.30%, and 0.34% respectively, which showed that the precision of the instrument was good.

Recovery
Test. 6 pieces of 1.00 g of known content of ASR were accurately weighted, and appropriate amount of each reference stock solution was added according to the  e results illustrated that the proposed method was of good accuracy.

Calculation of Relative Correction Factors (RCF).
e formula for calculating the relative correction factor is (f ) � (As/Cs)/(AR/CR). As is the peak area of the internal standard substance, AR is the peak area of the reference substance, Cs is the concentration of the internal standard substance, and CR is the concentration of the reference substance. e mixed reference solution prepared in Section 2.3 was injected into HPLC for analysis according to the chromatographic conditions under Section 2.5, respectively. Besides, the chromatographic peak areas of each component were recorded. e RCF of chlorogenic acid, senkyunolide I, senkyunolide H, coniferyl ferulate, senkyunolide A, ligustilide, and levistolide A was calculated with ferulic acid as internal standard. e results are shown in Table 2. e effect of three different chromatographic columns, different column temperatures, and different volumetric flows on RCF is shown in Supplementary Materials (Tables S1-S3). e RSD of each component was less than 3.00%, indicating that different chromatographic columns, different column temperatures, and different flow rates had no significant impact on the correction factors of each component, and the reproducibility was good.

Location of Chromatographic Peak.
According to the retention time obtained in "Section 3.2.1," the relative retention value of the components and the internal reference (r i/s � t Ri /t s ), where t Ri and t Rs are the retention time of the components to be tested and the internal standard ferulic acid, respectively) and their RSDs were calculated. e results showed that the RSD of the relative retention value of each component was less than 3%, indicating that the relative retention value was stable and can be used for the location of the chromatographic peak of the components to be tested. e results are shown in Table 3.

Sample Content Determination.
e calculation formula of QAMS is as follows: (CX) � f × A X /(A S '/C S '). AX is the peak area of the test article, C X is the concentration of the test article, A S ' is the peak area of the internal standard substance (ferulic acid), C S ' is the concentration of the internal standard substance (ferulic acid), and f is the RCF. e contents of each component in 6 batches of ASR and 4 kinds of its proprietary Chinese medicine were calculated by QAMS and external standard method (ESM), respectively. Each sample was determined three times. e results are shown in Table 4. It was found that the percentage difference (PD) PD � (QAMS − ESM)/[(QAMS + EMS)/2] × 100%) of these two methods was less than ±5.0%. e results of two determination methods had no significant differences, which

Conclusion
In this study, a QAMS method was established to determinate the contents of chlorogenic acid, senkyunolide I, senkyunolide H, coniferyl ferulate, senkyunolide A, ligustilide, and levistolide A with the ferulic acid as the internal standard substance. e results showed that there was no significant difference between QAMS and ESM by investigating different chromatographic columns, different column temperatures, and different flow rates; the results showed that the RSDs were all less than 3.00%, indicating that the change of chromatographic conditions had no significant effect on the relative correction factors of each component, and the reproduction was good. e abovementioned results indicate that the QAMS method that established could be accurately, economically, simply, and rapidly applied to the multicomponents analysis of ASR and its 12 kinds of preparations without reference substance. is work provided a scientific basis for quality control of ASR.

Data Availability
e data used to support the findings of this study are available from the corresponding authors upon request.