Development and Validation of a High-Performance Liquid Chromatography Method for Quality Assessment of Oriental Medicine, Dokhwalgisaeng-Tang

: Dokhwalgisaeng-tang (DHGST) is an herbal medicine formula that is frequently used in the treatment of arthritis in Korea and consists of 16 medicinal herbs. In this study, a simultaneous analysis method for quality assessment of DHGST by universal and widely used high-performance liquid chromatography was developed and validated. Twenty-four marker components were sep-arated on a reverse-phase SunFire C18 column (4.6 × 250 mm, particle size; 5 µ m) maintained at 40 ◦ C using a gradient elution of two mobile phase systems (0.1% aqueous formic acid and 0.1% formic acid in acetonitrile). The developed method was validated via linearity, limit of detection, limit of quantiﬁcation, recovery, and precision. Using the developed method, 24 marker components in DHGST were founded at 0.23–14.68 mg/g, and this method will be used as basic data for the quality assessment of DHGST or other herbal medicine prescriptions.

Many systematic reviews and meta-analyses have been reported for the use of DHGST in the treatment of rheumatoid arthritis, lumbar disk herniation, postmenopausal osteoporosis, and knee osteoarthritis [3][4][5][6][7], and research has been published by Liu et al. [8] on the effect of DHGST on stromal-cell-derived factor-1-induced inflammation and extracellular matrix degradation in human nucleus pulposus cells. It has also been reported that DHGST regulates autophagy and the P38/MAPK signaling pathway to prevent compression-induced matrix degradation and cell apoptosis in a rat model [9].
Methods for the quality control of DHGST based on high-performance liquid chromatography (HPLC) have been published by Chen et al. [26] and Wang et al. [27]; however, the analysis time in the former method was very long (500 min), and only four components (ferulic acid, osthole, gentiopicroside, and paeoniflorin) were detected. The method developed by Wang et al. [27] was based on only six components (chlorogenic acid, gentiopicrin, paeoniflorin, ferulic acid, glycyrrhizin, and osthole). Moreover, these studies focused on method efficacy rather than component analysis; thus, only a selection of component herbs (P. lactiflora, C. officinale, and G. uralensis) was examined, and no assay verification was performed. The development and validation of a simultaneous analysis method based on HPLC consistent quality evaluation of DHGST were therefore required and are described herein.

Plant Materials
The 16 raw herbal medicines used in this experiment are listed in Table S1; the plant names were confirmed on the website "The Plant List" (http://www.theplantlist.org/, accessed on 9 August 2021). These materials were purchased from Kwangmyungdag Medicinal Herbs (Ulsan, Korea). The origins of the raw herbal medicines were morphologically confirmed by Dr. Goya Choi, Korea Institute of Oriental Medicine (KIOM, Naju, Korea) according to guidelines and previous study protocols [28,29], and each material (2018-KE74-1 to 2018-KE74-16) was kept in KIOM.

DHGST Sample Preparation
DHGST powder extract was prepared according to a previously developed protocol [29]; the 16 herbal medicines were mixed in the weight ratio (w/w) shown in Table S1, then 50 L of distilled water was added, and the mixture was extracted at 100 • C for 2 h using an electric extractor. The extract solution was freeze-dried with an LP110R freeze-dryer (IlShinBioBase, Dongducheon, Korea) to obtain 1113.6 g (yield 22.3%) of a powder sample. The prepared DHGST sample was stored at −20 • C.

HPLC Simultaneous Quantification of the 24 Marker Compounds
Simultaneous quantification of the selected 24 marker analytes in the DHGST sample was conducted using a modification of a protocol developed in a previous study [29]. A Prominence LC-20A series (Shimadzu, Kyoto, Japan) HPLC instrument coupled with a photodiode array (PDA) detector capable of scanning the 190-800 nm region was used. The system was controlled and operated with LabSolution software (Ver. 5.53, SP3,Shimadzu, Kyoto, Japan). Full details of the analysis conditions are provided in Table S2.
A sample solution for simultaneous determination of the 24 marker analytes in the DHGST sample was prepared at a concentration of 10.0 mg/mL using 70% methanol, followed by ultrasonic extraction for 60 min. A standard solution of each reference standard compound was prepared at a concentration of 1.0 mg/mL using methanol and then stored in a refrigerator. All of the prepared solutions were filtered through a 0.2-µm membrane filter (Pall Life Sciences, Ann Arbor, MI, USA) before injection into the HPLC.

System Suitability Test of the Analytical Method
System suitability tests were conducted to evaluate the retention factor (k ), relative retention (α), resolution (Rs), number of theoretical plates (N), and tailing factor (Tf ) to ensure the adequate performance of the chromatography system for the developed method.

Method Validation of the Developed HPLC Analytical Assay
Validation of the analytical method developed in this study was performed with respect to linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision as described in the previous studies [29,30].

Selection of Marker Components for Quality Assessment of DHGST
As shown in Figure S2, we analyzed and compared the major components of each raw herbal medicine to select the marker compounds. All herbal medicines and components were scanned from 190 to 400 nm with a PDA detector during HPLC analysis using a mobile phase system of distilled-water-acetonitrile, with both phases containing 0.1% formic acid. Each marker component was confirmed by comparing its retention time and UV spectrum with those of the corresponding reference standard.
As shown in Figure S3, 40 main components in DHGST were analyzed using HPLC-PDA to enable the selection of the marker components. As a result, 24 components were identified as suitable markers in the DHGST sample, and these analytes were used as marker compounds for quality control of the DHGST sample in the subsequent studies.

System Suitability and Method Validation of the Developed HPLC Analytical Method
For efficient simultaneous analysis of markers in the DHGST sample, the suitability of the HPLC instrument was confirmed with respect to k′(1. 15-18. (Table S3). The regression equations for all the calibration curves showed excellent linearity, with a coefficient of determination (r 2 ) of 0.9999 to 1.0000 over the tested concentration range. The LOD and LOQ were calculated to be 0.004-0.061 μg/mL and 0.012-0.184 μg/mL, respectively (Table 1). Recovery was tested by adding three levels (low, medium, and high) of each standard solution to the DHGST sample, with results in the range of 95.47-102.81% ( Table 2). The repeatability  (18), cinnamaldehyde (19), glycyrrhizin (20), methyleugenol (21), safrole (22), decursin (23), and decursinol angelate (24).

System Suitability and Method Validation of the Developed HPLC Analytical Method
For efficient simultaneous analysis of markers in the DHGST sample, the suitability of the HPLC instrument was confirmed with respect to k (1. 15-18. (Table S3). The regression equations for all the calibration curves showed excellent linearity, with a coefficient of determination (r 2 ) of 0.9999 to 1.0000 over the tested concentration range. The LOD and LOQ were calculated to be 0.004-0.061 µg/mL and 0.012-0.184 µg/mL, respectively (Table 1). Recovery was tested by adding three levels (low, medium, and high) of each standard solution to the DHGST sample, with results in the range of 95.47-102.81% ( Table 2). The repeatability of the assay with respect to retention time and peak area was measured using a DHGST sample, and the RSD (%) was found to be ≤0.44% and ≤1.78%, respectively (Tables S4 and S5). Good RSD (%) values for intraday and interday precisions of ≤0.79% and ≤1.42%, respectively, were also recorded ( Table 3). Having validated the method as described, it was concluded that the current HPLC simultaneous analysis method is suitable for the quality assessment of DHGST samples. Table 1. Linear range, regression equation, coefficient of determination (r 2 ), limit of detection (LOD), and limit of quantification (LOQ) of 24 marker analytes for quantification by HPLC (n = 3).    Recovery (%) a = measured concentration/spiked concentration × 100%; RSD (%) b = standard deviation (SD)/mean × 100%.

Simultaneous Determination of 24 Markers Components for Quality Assessment of DHGST Sample
The established HPLC analysis method was successfully applied to the simultaneous determination of DHGST components for quality assessment. Simultaneous analysis of DHGST using the established assays showed that compounds 1-24 were present in 0.23-14.68 mg/g of freeze-dried sample (Table 4).