Liquid Chromatography Tandem Mass Spectrometry for the Simultaneous Quantiﬁcation of Eleven Phytochemical Constituents in Traditional Korean Medicine, Sogunjung Decoction

: The Sogunjung decoction (SGJD) is a traditional herbal formula that has been used to treat constipation and improve the constitution of inﬁrm children in Korea. In this study, simultaneous quantiﬁcation of gallic acid ( 1 ), magnoﬂorine ( 2 ), albiﬂorin ( 3 ), paeoniﬂorin ( 4 ), liquiritin apioside ( 5 ), liquiritin ( 6 ), liquiritigenin ( 7 ), coumarin ( 8 ), cinnamaldehyde ( 9 ), benzoylpaeoniﬂorin ( 10 ), and glycyrrhizin ( 11 ) was conducted using fast and sensitive liquid chromatography–tandem mass spectrometry (LC–MS) multiple-reaction monitoring to develop a quality-control protocol for the SGJD. A Waters Acquity UPLC BEH C 18 column (2.1 × 100 mm, 1.7 µ m) was used for the chromatographic separation of the 11 marker compounds in the SGJD using two mobile phases (5 mM ammonium acetate in distilled water containing 0.1% ( v/v ) formic acid, and acetonitrile). The MS parameters for a simultaneous analysis were capillary voltage (3.0 kV), source temperature (150 ◦ C), desolvation temperature (500 ◦ C), desolvation gas ﬂow (700 L/h), and cone gas ﬂow (50 L/h). The developed LC–MS method was validated by the evaluation of linearity, limits of detection, limits of quantiﬁcation, recovery and precision. By using the developed and validated assay, the 11 marker components in the SGJD were detected in amounts of 0.01–51.83 mg/g. positive and negative ion modes. Gallic acid ( 1 ), magnoﬂorine ( 2 ), albiﬂorin ( 3 ), paeoniﬂorin ( 4 ), liquiritin apioside ( 5 ), liquiritin ( 6 ), liquiritigenin ( 7 ), coumarin ( 8 ), cinnamaldehyde ( 9 ), benzoylpaeoniﬂorin ( 10 ) and glycyrrhizin 11 ).


Introduction
Traditional Korean medicine (TKM), traditional Chinese medicine (TCM), and Kampo medicine (KM) have been widely used in Asian countries such as Korea, China, and Japan to enhance human health and prevent various diseases. They are traditionally prescribed in the form of a combination of two or more herbal medicines based on a traditional medicine philosophy and have mostly been used as a decoction [1,2].
The Sogunjung decoction (SGJD) is a TKM called Xiao Jian Zhong Tang in China and Shokenchuto in Japan and was recorded for the first time in China in the Shanghanlun, a famous medical book of Chinese herbal medicine, written by Zhong-Jing Zhang in the third century during the Later Han Dynasty [3]. In addition, the SGJD is included in the Donguibogam, the definitive text of traditional Korean medicine, written by Jun Heo during the Joseon Dynasty and used to treat children's weakness, fatigue, chronic gastroenteritis, and bedwetting. The SGJD is a herbal formula made by mixing six medicinal herbs (Paeonia lactiflora Pallas, Cinnamomum cassia J. Presl, Glycyrrhiza uralensis Fischer, Zingiber officinale Roscoe, Zizyphus jujube Miller var. hoonensis T. B. Lee and Oryza sativa Linnè) in a defined ratio (7.5:4.5:1.5:1.0: 16:15.0) [4].
The SGJD has been reported to exhibit anti-allergy and immunomodulatory effects [5,6] and genotoxicity based on Ames and micronucleus tests [7]. Recently, clinical studies on the efficacy of SGJD granules for heartburn patients have been reported [8]. 2 of 10 Today, many analytical methods for quality control using high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS) have been reported for various kinds of compounds that are contained in herbal medicines or herbal prescriptions [9][10][11][12][13][14][15]. Recently, a simultaneous analysis of the major components of the SGJD was reported using HPLC coupled with a photodiode array detector (PDA) [16]. This study focused on simultaneous quantification for quality control of the SGJD using HPLC-PDA. However, studies using LC-MS have not been reported.

Plant Materials
As shown in Table S1, five raw plant materials (P. lactiflora, C. cassia, G. uralensis, Z. officinale and Z. jujube) were purchased in November 2017 from Kwangmyungdang Medicinal Herbs (Ulsan, Korea), a herbal medicine supplier for pharmaceuticals. The origin of these herbal medicines was confirmed by Dr. Seung-Yeol Oh based on The Dispensatory on the Visual and Organoleptic Examination of Herbal Medicine [17]. Specimens of the five raw herbs (2017KE63-1 to 2017KE63-5) were deposited at the Korea Institute of Oriental Medicine.

Preparation of SGJD Aqueous Extract
The SGJD was prepared from a formulation of the five raw herbs: P. lactiflora (2329.2 g), C. cassia (1397.5 g), G. uralensis (465.8 g), Z. officinale (310.6 g) and Z. jujube (496.9 g). Each herb was mixed and extracted according to the previously reported method [16]. The extract was freeze-dried to obtain an aqueous powder extract (887.1 g).

Preparation of Samples and Standard Solutions for LC-MS/MS MRM Analysis
A sample preparation for the quantification of compounds 1-11 in the SGJD by the LC-MS/MS MRM mode was prepared by adding 50 mL of 70% MeOH to 35.3 mg of a lyophilized and homogenized SGJD sample, followed by ultrasonic extraction with a Branson 8510 ultra-sonicator (Denbury, CT, USA) for 5 min at room temperature. The sample was then purified by vortexing for 1 min and filtering through a 0.2 µm hydrophobic filter. The filtrate was diluted 50 times with 70% MeOH and used for LC-MS/MS analysis.
Standard solutions of compounds 1-11 were made to a concentration of 100.0 µg/mL in MeOH, and stored in a refrigerator (ca. 4 • C) after preparation.

LC-MS/MS Equipment and Operating Conditions for Quantification of the Compounds 1-11
Quantification of compounds 1-11 in the SGJD was applied by modifying the analysis method that was developed in the previously study [18]. Briefly, LC-MS/MS analysis was performed with a Waters Acquity UPLC system (Milford, MA, USA) consisting of two pumps, a degasser, column oven and auto-sampler and a Waters Xevo TQ-XS triple quadrupole mass spectrometry system (Milford, MA, USA) coupled with an electrospray ionization (ESI) source operating in either negative or positive ion mode. Chromatographic separation of the 11 markers was performed with an Acquity UPLC BEH C 18 column

Validation of the LC-MS/MS Analysis Method
The developed LC-MS/MS MRM analysis method was validated by establishing linearity, range, limit of detection (LOD), limit of quantification (LOQ), accuracy, repeatability and intra-and inter-day precisions. The linearity was evaluated as the coefficient of determination (r 2 ) of the calibration curve prepared in different concentration ranges: 50.00-800.00 ng/mL for compound 1, 0.05-0.80 ng/mL for compound 2, 100.00-1600.00 ng/mL for compounds 3, 10, and 11, 250.00-4000.00 ng/mL for compound 4, 25.00-400.00 ng/mL for compound 5, 10.00-160.00 ng/mL for compounds 6 and 9, 1.00-16.00 ng/mL for compound 7 and 5.00-80.00 ng/mL for compound 8. LOD and LOQ were calculated based on signal-to-noise ratios of 3 and 10, respectively.
The accuracy of the developed method was evaluated based on extraction recovery tests. Briefly, the recovery (%) was measured by adding a standard compound (low, medium, and high) of a known concentration to the SGJD sample and calculated from Equation (1): To evaluate the precision of the developed LC-MS/MS MRM analysis method, repeatability and intra-and inter-day precisions were measured, and the relative standard deviation (RSD) value of each precision was measured. The repeatability of the method was established by repeated measurements of a mixed standard solution, and the RSD values of the retention time and peak area of each compound were calculated. Intra-and inter-day precision was established by calculating the RSD of the analysis of a mixed standard solution on one day and on three consecutive days, respectively. The RSD was calculated by Equation (2):

Optimization of LC-MS/MS Conditions
The analysis conditions for the LC-MS/MS MRM method were optimized for the simultaneous quantification of the 11 marker components for quality control of the SGJD. The optimal analysis was established with an Acquity UPLC BEH C 18 column (2.1 × 100 mm, 1.7 µm), a column temperature of 45 • C, two mobile-phase system (0.1% (v/v) FA and 5 mM AF in DW, and ACN) and a flow rate of 0.3 mL/min, respectively. By using the optimized LC-MS/MS MRM method, compounds 1-11 were detected within 6 min ( Figure 1, Table 1). 50.00-800.00 ng/mL for compound 1, 0.05-0.80 ng/mL for compound 2, 100.00-1600.00 ng/mL for compounds 3, 10, and 11, 250.00-4000.00 ng/mL for compound 4, 25.00-400.00 ng/mL for compound 5, 10.00-160.00 ng/mL for compounds 6 and 9, 1.00-16.00 ng/mL for compound 7 and 5.00-80.00 ng/mL for compound 8. LOD and LOQ were calculated based on signal-to-noise ratios of 3 and 10, respectively. The accuracy of the developed method was evaluated based on extraction recovery tests. Briefly, the recovery (%) was measured by adding a standard compound (low, medium, and high) of a known concentration to the SGJD sample and calculated from Equation (1): To evaluate the precision of the developed LC-MS/MS MRM analysis method, repeatability and intra-and inter-day precisions were measured, and the relative standard deviation (RSD) value of each precision was measured. The repeatability of the method was established by repeated measurements of a mixed standard solution, and the RSD values of the retention time and peak area of each compound were calculated. Intra-and inter-day precision was established by calculating the RSD of the analysis of a mixed standard solution on one day and on three consecutive days, respectively. The RSD was calculated by Equation (2):

Optimization of LC-MS/MS Conditions
The analysis conditions for the LC-MS/MS MRM method were optimized for the simultaneous quantification of the 11 marker components for quality control of the SGJD. The optimal analysis was established with an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm), a column temperature of 45 °C, two mobile-phase system (0.1% (v/v) FA and 5 mM AF in DW, and ACN) and a flow rate of 0.3 mL/min, respectively. By using the optimized LC-MS/MS MRM method, compounds 1-11 were detected within 6 min (Figure 1, Table 1).

Identification of Compounds 1-11 for LC-MS/MS MRM Assay
The  Table 1). Under the optimized LC-MS/MS MRM conditions as shown in Table  1, the precursor ion (Q1) and product ion (Q3) of each marker compound were set and analyzed. The

Identification of Compounds 1-11 for LC-MS/MS MRM Assay
The LC-MS/MS analysis of compounds 1-11 in MRM mode was performed using the ESI source (negative and positive ion modes). Precursor ions for compounds 1, 4-6, 9 and  Table 1). Under the optimized LC-MS/MS MRM conditions as shown in Table  1, the precursor ion (Q1) and product ion (Q3) of each marker compound were set and analyzed. The  [26]. The Q1 and Q3 spectra of compounds 1-11 are shown in Figure S2.

Method Validation of the LC-MS/MS MRM Assay
The LC-MS/MS analysis developed in this study was verified with respect to linearity, LOD, LOQ, accuracy and precision. As shown in Table 2, the r 2 values of all compounds were >0.99, which showed that the linearity was good, and the LOD and LOQ values were also calculated as 0.004-32.468 ng/mL and 0.013-97.403 ng/mL, respectively. The extraction recovery test for evaluation of the accuracy of the analytical method calculated from Equation (1) (see Section 2.6) was 96.43-107.60 (Table 3). In the verification of repeatability and intra-and inter-day precisions, the RSD (%) values of compounds 1-11 were less than 9.00%, showing good precision (Table 4). These results suggest that the developed method is an appropriate analysis method for quality control of the SGJD. Table 2. Linear range, regression equation, coefficient of determination (r 2 ), limit of detection (LOD), and limit of quantification (LOQ) for LC-MS/MS MRM analysis of compounds 1-11 (n = 3).

Analyte
Linear

Quantification of Compounds 1-11 in SGJD Samples by LC-MS/MS MRM Analysis
The developed and validated LC-MS/MS analysis method was applied for quantitative analysis of compounds 1-11 in the SGJD. The retention times of compounds 1-11 in the sample were 1.01, 1.31, 1.40, 1.45, 1.53, 1.65, 2.99, 3.06, 2.31, 4.42 and 5.20 min, respectively ( Figure 1 and Figure S3). As shown in Table 1, an analysis of the SGJD under the optimized MRM conditions for each compound showed that compounds 1-11 were present in the sample in amounts of 0.01-51.83 mg/g. Among these markers, compounds

Conclusions
In this study, a sensitive, accurate, and fast LC-MS/MS MRM analysis method for quality control of the SGJD was first developed using the selected compounds 1-11. The method was verified by assessing the linearity, LOD, LOQ, accuracy, and precision parameters, and was successfully applied to sample analysis. The proposed LC-MS/MS MRM assay will be useful for the quality evaluation of the SGJD and for further research on other TKM, TCM, and KM along with the HPLC-PDA assay.

Data Availability Statement:
The data presented in this study are available in the article (Tables  and Figures).

Conflicts of Interest:
The authors declare no conflict of interest.