HPLC–PDA and LC–MS / MS Analysis for the Simultaneous Quantiﬁcation of the 14 Marker Components in Sojadodamgangki-Tang

: Sojadodamgangki-tang (SDGT) is a traditional Korean medicine consisting of 12 medicinal herbs that has been used in Korea for the treatment of asthma since ancient times. However, the quality control of herbal formulas that contain two or more herbal medicines remains challenging. In this study, 14 marker components were analyzed simultaneously by using high-performance liquid chromatography with photodiode array detection in addition to the use of liquid chromatography–tandem mass spectrometry for quality evaluation of SDGT. The simultaneous determination of the 14 marker components was validated in terms of linearity, recovery, and precision. The established methods can provide useful data for the quality control of SDGT and related herbal formulas.


Introduction
Traditional Chinese medicine (TCM), traditional Korean medicine (TKM), and kampo medicine (KM) have long been used in clinical practice for the treatment or prevention of various diseases in countries such as Korea, China, and Japan. The treatments are generally regarded as safe and have become standardized over centuries of refinement. However, with the development of modern science, research on these herbal prescriptions has become more frequent. Moreover, there is an increasing demand for regulatory documentation to certify the safety and standardization of TCM, TKM, and KM, and validated methods are therefore required.
Sojadodamgangki-tang (SDGT) is a TKM consisting of 12 herbal medicines that has been used to treat asthma [1]. The effects of SDGT on SO 2 -induced respiratory injury, type I and type IV allergic reactions, and allergic asthma have been reported [2][3][4][5].

Plant Materials
The 12 component herbs of SDGT (Table S1) were purchased from the herbal medicine manufacturer Kwangmyungdag Medicinal Herbs (KMH, Ulsan, Korea) in November 2017. The origin of these raw materials was confirmed by herbalist, Seung Yeol Oh, CEO of KMH (Ulsan, Korea), based on "The Dispensatory on the Visual and Organoleptic Examination of Herbal Medicine" [21]. A voucher specimen (from 2018-CA01-1 to 2018-CA01-12) has been deposited at the Herbal Medicine Research Division, Korea Institute of Oriental Medicine (KIOM).

Chemicals and Reagents
The 14 reference standard compounds ( Figure S1) used for the qualitative and quantitative analysis of SDGT were purchased from the following natural product suppliers: LIQA ( HPLC-grade reagents (methanol, acetonitrile, and water) and formic acid (FA, for HPLC) were purchased from J.T.Baker (Phillipsburg, NJ, USA) and Merck KGaA (Darmstadt, Germany), respectively.

Preparation of SDGT Water Extract
As demonstrated in Table 1, the 12 crude herbs that make up SDGT were combined, 50 L of distilled water was added, and the mixture was extracted under pressure (98 kPa) at 100 • C for 2 h using an electric extractor (COSMOS-660; Kyungseo E&P, Incheon, Korea). The extracted SDGT aqueous solution was filtered using a standard sieve (53 µm mesh) and then lyophilized to obtain 635.3 g (yield, 12.71%) of powder extract. Table 1. Linear range, regression equation, r 2 , limit of detection (LODs), and limit of quantification (LOQs) for marker compounds using high-performance liquid chromatography-photodiode array detection (HPLC-PDA) (n = 3).

Compound
Linear

Preparations of Sample and Standard Stock Solutions for HPLC Analysis
In order to simultaneously determine the 14 marker components (LIQA, LIQ, NOD, NAR, NARG, HES, NPON, PON, API, GLY, HON, DEC, DECA, and PRAA) in the SDGT aqueous decoction, 10 mL of 70% methanol was added to 100 mg of the lyophilized SDGT sample, followed by ultrasonic extraction via a Branson 8510 (Denbury, CT, USA) ultra-sonicator. The extract was filtered through a 0.2 µm membrane filter (Pall Life Sciences, Ann Arbor, MI, USA) and injected into the HPLC system.
Standard stock solutions of the 14 reference standard compounds were prepared at a concentration of 1000.0 µg/mL using methanol whilst the prepared stock solutions were stored in a refrigerator until required.

Validation of the HPLC Analytical Procedure
Generally, to apply the developed method for the simultaneous quantitative analysis and standardization of TKM formulations, the method must be validated. In this study, we validated the analytical procedure in accordance with the following strict parameters: limit of detection (LOD), limit of quantification (LOQ), and accuracy, and precision according to the International Conference on Harmonisation (ICH) guidelines [22].
In short, the linearity of these parameters was confirmed by the value of the coefficient determination (r 2 ) of the calibration curve, drawn at different concentration ranges of each marker Appl. Sci. 2020, 10, 2804 4 of 11 compound. LOD and LOQ concentrations were set using equations where σ is the standard deviation of the y-intercept and S is the slope of the calibration curve.
The accuracy was evaluated based on the recovery test of the standard addition method, using the equation: where precision was verified by intraday and interday precision and repeatability measurements. Intraday and interday precisions were measured five times in a single day and on three consecutive days at different concentrations (low, medium, and high) using the standard solution. The relative standard deviation (RSD) of the recorded series of repeatability was also measured six times using the equation: The system suitability of the assay was evaluated using the parameters such as capacity factor (k'), selectivity factor (α), resolution (Rs), number of theoretical plates (N), and tailing factor (Tf ).  Table S2. Other parameters were: capillary voltage, 1.2 kV; source temperature, 150 • C; desolvation temperature, 450 • C; desolvation gas flow, 800 L/h; cone gas flow, 50 L/h. All data were processed using Waters MassLynx software (Version 4.2; Waters, Milford, MA, USA).

Method Validation of the HPLC Analytical Method
System characteristics such as capacity factor (k), selectivity (α), theoretical plate number (N), resolution (Rs), and tailing factor (Tf), were all evaluated to ensure that the complete system consisting of the analytical instrument, analytical operation, and samples to be analyzed was performing as required. The results (Table S3) confirmed that all parameters were suitable for the method. As shown in Table 1, the r 2 values of all the components showed excellent linearity of ≥0.99996, and LOD and LOQ concentrations were calculated to be 0.01-0.37 µg/mL and 0.04-1.11 µg/mL, respectively. The extraction recovery (%) for the evaluation of each marker component's accuracy at three different concentrations was determined to be 95.89%-104.18% and the RSD value was calculated to be less than 2.50% (Table 2). Intraday, interday, and repeatability precisions showed an RSD (%) of less than 3.00% (Table 3). This result confirmed the validity of the established simultaneous analysis method for the quality assessment of SDGT.

Method Validation of the HPLC Analytical Method
System characteristics such as capacity factor (k), selectivity (α), theoretical plate number (N), resolution (Rs), and tailing factor (Tf ), were all evaluated to ensure that the complete system consisting of the analytical instrument, analytical operation, and samples to be analyzed was performing as required. The results (Table S3) confirmed that all parameters were suitable for the method. As shown in Table 1, the r 2 values of all the components showed excellent linearity of ≥0.99996, and LOD and LOQ concentrations were calculated to be 0.01-0.37 µg/mL and 0.04-1.11 µg/mL, respectively. The extraction recovery (%) for the evaluation of each marker component's accuracy at three different concentrations was determined to be 95.89-104.18% and the RSD value was calculated to be less than 2.50% (Table 2). Intraday, interday, and repeatability precisions showed an RSD (%) of less than 3.00% (Table 3). This result confirmed the validity of the established simultaneous analysis method for the quality assessment of SDGT.

LC-MS/MS Confirmation
As shown in Table S2, all marker components in the LC-MS system were identified from their molecular ion peaks in the positive or negative ion modes. Six analytes (LIQA, LIQ, NAR, NARG, GLY, and HON) were detected using the negative ion mode  (Table  S4). The LOD and LOQ values of each component were 0.001-0.710 ng/mL and 0.003-2.131 ng/mL, respectively, based on signal-to-noise ratios of 3.3 and 10. (Table S4).  (Table 4). Of these markers, PON, the marker component of Ponciri Fructus Immaturus, was found to be the most abundant in SDGT (5.20-5.25 mg/g).    9.74, 9.91, 9.96, and 10.14 min (Figure 2 and Figure S2), respectively, and the concentrations of these analytes were found to be 0.005-0.384 mg/g (Table 5).

Conclusions
In this study, both qualitative and quantitative analysis for the quality control of SDGT using HPLC-PDA and LC-MS/MS were developed and validated for the first time. Simultaneous analysis of the 14 marker components in SDGT via the use of the two analytical methods was also successfully performed and validated. These newly validated and established HPLC-PDA and LC-MS/MS methods are expected to be effective protocols for the quality control of SDGT and related herbal formulas going forward.

Conclusions
In this study, both qualitative and quantitative analysis for the quality control of SDGT using HPLC-PDA and LC-MS/MS were developed and validated for the first time. Simultaneous analysis of the 14 marker components in SDGT via the use of the two analytical methods was also successfully performed and validated. These newly validated and established HPLC-PDA and LC-MS/MS methods are expected to be effective protocols for the quality control of SDGT and related herbal formulas going forward.

Conflicts of Interest:
The authors have declared no conflict of interest.