Simultaneous Analysis of 19 Marker Components for Quality Control of Oncheong-Eum Using HPLC–DAD

Oncheong-eum (OCE) is a traditional herbal prescription made by combining Samul-tang and Hwangryunhaedok-tang. It is primarily used to treat gynecological disorders such as metrorrhagia and metrostaxis. In the present study, we focused on developing and validating a simultaneous assay for the quality control of OCE using 19 marker components (gallic acid, 5-(hydroxymethyl)furfural, chlorogenic acid, geniposide, coptisine chloride, jatrorrhizine chloride, paeoniflorin, berberine chloride, palmatine chloride, ferulic acid, nodakenin, benzoic acid, baicalin, benzoylpaeoniflorin, wogonoside, baicalein, wogonin, decursin, and decursinol angelate). This analysis was performed using high-performance liquid chromatography coupled with a diode array detector, and chromatographic separation of the 19 markers was carried out using a SunFireTM C18 reversed-phase column and gradient elution conditions with two mobile phases (0.1% aqueous formic acid–0.1% formic acid in acetonitrile). The developed analytical method was validated through linearity, limits of detection and quantification, recovery, and precision. Under this assay, 19 markers in OCE samples were detected at not detected–9.62 mg/g. The analytical methods developed and validated in our research will have value as basic data for the quality control of related traditional herbal prescriptions as well as OCE.


Introduction
In general, traditional herbal prescriptions consist of two or more herbal medicines. They have been used for a long time in Asian countries, especially Korea, China, and Japan, because of the advantages of fewer side effects and multicomponent/multitarget compared with synthetic drugs or Western medicine [1][2][3]. However, despite their prolonged use, modern scientific validation of their biological activity and research on standardization to maintain the consistency of raw materials are still needed.
Oncheong-eum (OCE; "Wen-Qing-Yin" in Chinese and "Unsei-in" in Japanese), also called "Haedoksamultang", is a traditional herbal prescription that combines Samul-tang and Hwangryunhaedok-tang and consists of eight herbal medicines: Angelica gigas Nakai, Cnidium officinale Makino, Paeonia lactiflora Pall., Rehmannia glutinosa (Gaertn.) DC., Coptis chinensis Franch., Phellodendron chinensis C.K.Schneid., Scutellaria baicalensis Georgi, and Gardenia jasminoides Ellis, in the same weight ratio [4]. Since OCE was first recorded in the Manbyeonghoichun of Gong Tingxian, a physician in the Ming Dynasty, it has also been transmitted in Dongeuibogam of Heo Jun, a famous medical book of the Joseon Dynasty [4,5]. This prescription has been used to stop abnormal uterine bleeding, remove fever, and treat stomach pain. It is also used for skin diseases and metabolic diseases [5,6].
Various biological activities of OCE have been reported, such as the inhibition of cell proliferation in the human hepatocarcinoma cell line [7,8], inhibition of melanogenesis and tyrosinase activity in the murine melanoma cell line, skin regeneration and wrinkle improvement in the human normal fibroblast cell line [9], and pruritus [10,11].

Identification of the Major Components of Each Herbal Medicine Constituting OCE
To develop a simultaneous analysis method for the quality control of OCE using HPLC-DAD, the primary ingredients contained in eight raw herbal medicines were first explored. The ingredients selected for analysis in each raw herb are as follows: chlorogenic acid, nodakenin, decursin, and decursinol angelate from A. gigas; ferulic acid from C. officinale; albiflorin, paeoniflorin, benzoic acid, gallic acid, and benzoylpaeoniflorin from P. lactiflora; 5-(hydroxymethyl)furfural from R. glutinosa; berberine chloride, coptisine chloride, jatrorrhizine chloride, and palmatine chloride from C. japonica and P. chinensis; baicalin, baicalein, wogonin, and wogonoside from S. baicalensis; and geniposide and gardenoside from G. jasminoides. As a result of a comparative analysis of each constituent herbal medicine and their main components in HPLC using a reversed-phased C 18 column and a distilled water (DW)-acetonitrile (ACN) mobile system (both containing 0.1% formic acid; FA), it was confirmed that the target components were present in each constituent herbal medicine ( Figure S1).

Selection of Marker Analytes for Quality Control of OCE Using an HPLC Sytem
After performing as in Section 3.1 and performing the same in the OCE sample, 19 compounds were detected, whereas two components, gardenoside, and albiflorin, were not detected ( Figure S2A,B). Of these two components, gardenoside showed a peak at the same retention time as the standard compound, but as a result of UV spectrum comparison, it was identified as a different component and thus excluded from analysis ( Figure S2C). In addition, albiflorin was detected in the sample, but separation from other neighboring components was not complete, so it was not selected as a marker component. Finally, among the 21 components, 19 compounds completely isolated and identified from the OCE sample were selected as marker components suitable for the quality control of OCE. The chemical structures of compounds 1-19 selected as marker analytes for the quality control in OCE are shown in Figure S3.

Optimization of HPLC Analysis Conditions
We compared several conditions to search for efficient chromatographic separation conditions for the 19 marker analytes selected to achieve the development of an optimal HPLC simultaneous method for the quality control of OCE. First, the resolution of markers was compared by comparing reversed-phase C 18 columns (4.6 mm × 250 mm, 5 µm) such as SunFire TM (Waters, Milford, MA, USA), Gemini (Phenomenex, Torrance, CA, USA), Capcellpak UG80 (Shiseido, Tokyo, Japan), and Hypersil GOLD (Thermo Fisher Scientific Inc., San Jose, CA, USA). Next, the types of acid added to the mobile phase (FA, trifluoroacetic acid; TFA, acetic acid; AA, and phosphoric acid; PA), and the temperatures of the column oven (30, 35, 40, and 45 • C) were also compared. Figure 1 shows the representative HPLC chromatogram measured under the optimal analysis conditions finally selected (SunFire TM C 18 column, column temperature of 40 • C, and mobile phase system of 0.1% FA in DW-0.1% FA in ACN, based on the comparison and search conditions). All analytes were eluted within 65 min without the influence of neighboring peaks with a resolution of 16.9 or higher (Table S3).  Figure S3.

System Suitability and Stability Tests
The system suitability values were 1.17-21.22 (capacity factor; k ), 1.01-1.75 (selectivity factor; α), 18,808.16-1,737,667.55 (number of theoretical plates; N), 1.69-28.98 (resolution; Rs), and 1.01-1.18 (tailing factor; Tf ). furthermore, the stability of each marker analyte measured using a standard solution for three days (0, 24, and 48 h) showed an RSD value in the range of 0.68% to 2.36%. The detailed results for each marker component are summarized in Table S4.

Plant Materials
The eight raw herbal ingredients shown in Table S1 were purchased from Kwangmyungdang Pharmaceutical (Ulsan, Korea), a specialized herbal medicine manufacturing company, in November 2017. Each herbal medicine was used after morphological identification according to the guideline "The Dispensatory on the Visual and Organoleptic Examination of Herbal Medicine" by Dr. Goya Choi, Korea Institute of Oriental Medicine (KIOM, Daejeon, Korea) [26]. Each herbal medicine was deposited at the KM Science Research Division, KIOM (specimen no.: from 2018KE81-1 to 2018KE81-8).

Chemicals and Reagents
All reference standard compounds used for HPLC analysis were purchased from standard compound manufacturers: compounds 1 (CAS No. 149-91-7

Preparation of OCE Water Extract
OCE water extract (OCE-1) was prepared at KIOM according to a previously reported manufacturing method [27,28]. That is, after mixing the same amount (each 625.0 g) of the eight herbal medicines shown in Table S1, 50 L of DW was added, and the mixture was extracted for 2 h at 100 • C using an electric extractor, COSMOS-660 (Kyungseo E&P, Incheon, Korea). According to the previously reported manufacturing method. Subsequently, the extracted water solution was filtered using a sieve (53 µm mesh). As a final step for producing a powder sample, the filtered extract was freeze-dried using an LP100R freeze dryer (IlShinBioBase, Yangju, Korea) (1232.6 g, yield 24.7%). Apart from the sample prepared by KIOM, the other four commercial samples (from OCE-2 to OCE-5) were purchased from different pharmaceutical companies, Kyungbang (Incheon, Korea), Jungwoo Medicines (Asan, Korea), Hankookshinyak (Nonsan, Korea), and Tsumura & Co. (Tokyo, Japan), respectively.

Preparation of Test Solutions and Standard Solutions for HPLC-DAD Analysis
For the test solutions for simultaneous determination of the 19 markers in OCE, about 100 mg was accurately taken of each prepared OCE water extract and commercially available products in a 10 mL volumetric flask, then filled with 70% MeOH (100 mg/10 mL). The continuously mixed samples were subjected to ultrasonic extraction at room temperature for 60 min. For the quantitative analysis of compounds 4, 7, 8, and 13, the prepared test solution was diluted 10-fold and used. All solutions were filtered before analysis using a 0.2 µm syringe filter (Pall Life Sciences, Ann Arbor, MI, USA) and then injected into the HPLC instrument.
Standard solutions of the 19 reference standard compounds were prepared at a concentration of 1.0 mg/mL using methanol or DMSO-MeOH solution (1:1), and then used while refrigerated.

HPLC Instrument and Analysis Conditions
The HPLC instrument used for simultaneous analysis of the 19 markers in OCE was a Prominence LC-20A modular system (Shimadzu Co., Tokyo, Japan) consisting of a quaternary pump (LC-20AT), DAD (SPD-M20A), autosampler (SIL-20A), and column oven (CTO-20A). The system is operated and controlled by LabSolution software (Version 5.53, SP3, Kyoto, Japan). Chromatographic separation for simultaneous analysis of marker analytes was performed using a reversed-phase column, SunFire TM C 18 column (4.6 mm × 250 mm, 5 µm, Waters, Milford, MA, USA), and gradient elution with two mobile phases (0.1% FA in DW-0.1% FA in ACN). Other detailed analysis conditions, including gradient elution conditions, are shown in Table S2.

Validation of the Developed HPLC Analytical Method
The analytical HPLC method developed to be applied to the quality control of OCE was validated by measuring and confirming various parameters such as linearity, LOD, LOQ, recovery, and precision [29]. First, the linearity of each marker component was checked in the following concentration ranges (0.31-20.00 µg/mL for compounds 1, 6, 10, 12-14, and 17-19; 0.47-30.00 µg/mL for compounds 2-4, 7, 8, and 11; 0.78-50.00 µg/mL for compounds 5, 9, 15, and 16) and evaluated through the r 2 of the prepared calibration curve.
Second, LOD and LOQ were calculated by the following equations, respectively. LOD = 3.3 × σ/S and LOD = 10 × σ/S, where σ and S represent the standard deviation of the y-intercept and the slope of the calibration curve in the regression equation of each marker measured three times, respectively.
Third, the recovery was validated by the standard addition method. Briefly, after accurately taking a 100 mg OCE powder sample in a 10 mL volumetric flask, three concentration levels (low, medium, and high) of each known marker compound were added. After that, the pretreatment process was the same as the preparation of the sample solution in Section 2.4. Extraction recovery (%) was calculated using following equation: Recovery (%) = found amount/spiked amount × 100.
Finally, the precision was evaluated as the relative standard deviation (RSD) of intraday and interday precisions and repeatability. The intraday and interday precisions were evaluated by calculating the RSD after measuring five times each for one day and three consecutive days using a standard solution of three concentrations in which the 19 markers were mixed. Repeatability was evaluated by obtaining the RSD of each marker analyte's retention time and peak area after six repeated measurements.

System Suitability and Stability Tests
The system suitability test was validated by evaluating the k , α, Rs, N, and Tf to evaluate the normal operation of the analysis system [27]. Furthermore, the stability of each marker component was tested for three days (0, 24, and 48 h) at 21 ± 1 • C using a standard solution.

Conclusions
In this study, a simultaneous analysis method using convenient, accurate, and reproducible HPLC-DAD for the quality control of OCE was developed and validated. This analytical method has been successfully applied to qualitative and quantitative analysis