Simultaneous Analysis to Evaluate the Quality of Insamyangpye–Tang Using High-Performance Liquid Chromatography–Photo Diode Array Detection

Insamyangpye–tang (ISYPT) is a traditional medicinal formula comprised of 13 herbs and has been used in East Asia to treat lung-related diseases. However, to our knowledge, no method of analysis for its quality control has been reported. In this study, a method of analysis for quality control of ISYPT was developed using high-performance liquid chromatography. Chromatographic separation, analysis, and assay verification were performed with a distilled water–acetonitrile mobile phase system, both containing 0.1% (v/v) trifluoroacetic acid, and a Gemini C18 analytical column (4.6 mm × 250 mm, 5 μm) using authentic standards for eight marker compounds. These marker constituents were detected simultaneously at 0.09–5.95 mg/g. The analysis method developed can be used for basic quality control of ISYPT.


Introduction
There is a growing interest and demand for traditional Korean medicine (TKM), traditional Chinese medicine (TCM), and Kampo medicine (KM) worldwide, especially in East Asian countries. In general, they are composed of two or more different herbal medicines and have both multi-component and multi-target features. Because they are known to have few side effects, traditional medicines have the advantage in that they can be used widely to treat and prevent various diseases [1][2][3]. To manage the quality of TKM, TCM, and KM for their continued use, standardization studies are required.
In the present study, for the first time to our knowledge, a method of analysis for quality control of ISYPT to assure consistency was developed using a high-performance liquid chromatography-photodiode array detection (HPLC-PDA) system for simultaneous measurement of eight marker compounds. The eight marker compounds selected were mulberroside A (MBRSA) as found in M. alba, liquiritin apioside (LQRTA), liquiritoside (LQRTS), and glycyrrhizic acid (GCRZA) as found in G. uralensis, naringin (NRG), didymin (DDM), and poncirin (PCR) as found in P. trifoliate, and schizandrol A (SZDRA) as found in S. chinensis.

Plant Materials
The 13 types of medicinal herbs that make up ISYPT, as shown in Table S1, were obtained from a specialized medicinal herb store, Kwangmyungdag Medicinal Herbs (Ulsan, Korea), in 2018. All of them were evaluated morphologically by Dr. Goya Choi, Herbal Medicine Resources Research Center (HMRRC), Korea Institute of Oriental Medicine (KIOM, Naju, Korea), according to National Institute of Food and Drug Safety Evaluation guidelines, the Korean Pharmacopoeia, and the Korean Herbal Pharmacopoeia [17][18][19]. Samples of each (2018-CA03-1 to 2018-CA03-13) have been deposited in the KIOM herbarium.

ISYPT Extraction to Produce a Sample
To prepare an ISYPT water extract, the constituent medicinal herbs were mixed in the weight ratio (g, w/w) shown in Table S1, and then 50 L of water was added for extraction at 100 • C for 2 h. Then, the solution of extract was freeze-dried to produce a powdered sample (988.8 g). The ISYPT sample thus prepared was stored at −20 • C until use.

Preparation of Sample Solution and Standard Stock Solution
Quantification for quality control of the ISYPT using the marker compounds (MBRSA, LQRTA, LQRTS, NRG, DDM, PCR, GCRZA, and SZDRA) was performed by HPLC-PDA. As a sample, 100 mg of lyophilized ISYPT was placed in a 10-mL volumetric flask, and DW and 70% MeOH were added, respectively. Then, each mixture was sonicated for 60 min and filtered using a 0.2 µm polypropylene membrane filter (Pall Life Sciences, Ann Arbor, MI, USA) before analysis.
A stock solution was prepared using each authentic standard at 1000 µg/mL in MeOH and then stored at −4 • C until use.

HPLC Analysis for ISYPT Quality Assessment
Simultaneous analysis of ISYPT constituents was conducted using previous analytical protocols [20]. In brief, the HPLC system used in this analysis was a Shimadzu Prominence LC-20A series (Kyoto, Japan), and PDA was used to detect the constituents.
LCSolution software (version 1.24, SP1) was used to acquire and process data. Detailed parameters for HPLC for the quantitative analyses are shown in Table S2.

System Suitability
To develop a method for simultaneous analysis of marker compounds, system suitability factors, capacity factor (k ), relative retention (α), resolution (Rs), number of theoretical plates (N), and tailing factor (Tf ), were identified.

Validation of the HPLC Analytical Method
Validation (linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision) of the HPLC method developed to assess the quality of ISYPT samples was conducted in the same manner as in a previous study [20] and according to the International Conference on Harmonisation guidelines [21].

Selection of Compounds to Evaluate the Quality of ISYPT
We detected eight marker compounds after HPLC of ISYPT samples containing the 23 constituents identified in Section 3.1 ( Figure S3). After determining eight marker compounds for ISYPT ( Figure S1), further studies such as establishing of simultaneous analysis method, method validation, and quantification were conducted.

Validation of the HPLC Analytical Method
To analyze the stability of the HPLC method, system suitability for parameters such as k , α, N, Rs, and Tf was tested using a solution of authentic standards. As shown in Table S3, as a result of considering all parameters, the method is suitable for simultaneous analysis of the chosen standards. The coefficients of determination (r 2 ), which are indicators of linearity, were all 0.9999, showing good linearity ( Table 1). The LOD 0.03-0.41 µg/mL and LOQ 0.08-1.21 µg/mL (Table 1) were calculated in the same way as they were in previous studies [15,16]. As shown in Table 2, the extraction recovery test performed to verify accuracy was 95.67-102.38% in samples to which authentic standards were added at various concentrations (low, medium, and high) to 103.50%, and relative standard deviation (RSD) (%) was 0.23-2.35%. Repeatability was expressed as RSD (%) after six repeated measurements using a solution of the standards. All authentic standards had <1.0% RSD for retention time and peak area, showing good repeatability (Table 3 and Tables  S4 and S5). The intra-(one day) and interday (three consecutive days) precision also showed good results with RSD (%) <4.0% (Table 3). Based on verification results indicated above, the method for quality control of ISYPT developed in the present study is acceptable.

Validation of the HPLC Analytical Method
To analyze the stability of the HPLC method, system suitability for parameters such as k′, α, N, Rs, and Tf was tested using a solution of authentic standards. As shown in Table S3, as a result of considering all parameters, the method is suitable for simultaneous analysis of the chosen standards. The coefficients of determination (r 2 ), which are indicators of linearity, were all 0.9999, showing good linearity ( Table 1). The LOD 0.03-0.41 μg/mL and LOQ 0.08-1.21 μg/mL (Table 1) were calculated in the same way as they were in previous studies [15,16]. As shown in Table 2, the extraction recovery test performed to

Simultaneous Quantification of the Eight Marker Constituents of ISYPT
The HPLC method newly developed and verified in the present study was applied to the simultaneous analysis of eight marker constituents in lyophilized ISYPT. The constituents were quantified using a full scan from 190 to 400 nm with PDA detector, and each constituent was quantified based on its maximum UV absorption as follows: 250 nm for GCRZA and SZDRA, 275 nm for LQRTA and LQRTS, 280 nm for NRG, DDM, and PCR, and 325 nm for MBRSA. The eight representative marker constituents (MBRSA, LQRTA, LQRTS, NRG, DDM, PCR, GCRZA, and SZDRA) were detected in the lyophilized ISYPT samples at concentrations ranging 0.09-5.95 mg/g (Table 4).