Simultaneous Analysis for Quality Control of Traditional Herbal Medicine, Gungha-Tang, Using Liquid Chromatography–Tandem Mass Spectrometry

Gungha-tang (GHT), a traditional herbal medicine, consists of nine medicinal herbs (Cnidii Rhizoma, Pinelliae Tuber, Poria Sclerotium, Citri Unshius Pericarpium, Citri Unshius Pericarpium Immaturus, Aurantii Fructus Immaturus, Atracylodis Rhizoma Alba, Glycyrrhizae Radix et Rhizoma, and Zingiberis Rhizoma Recens). It has been used for various diseases caused by phlegm. This study aimed to develop and verify the simultaneous liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis method, using nine marker components (liquiritin apioside, neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, liquiritigenin, glycyrrhizin, and 6-shogaol) for quality control of GHT. LC–MS/MS analysis was conducted using a Waters TQ-XS system. All marker analytes were separated on a Waters Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) using gradient elution with a distilled water solution (containing 5 mM ammonium formate and 0.1% [v/v] formic acid)–acetonitrile mobile phase. LC–MS/MS multiple reaction monitoring (MRM) analysis was carried out in negative and positive ion modes of an electrospray ionization source. The developed LC–MS/MS MRM method was validated by examining the linearity, limits of detection and quantification, recovery, and precision. LOD and LOQ values of nine markers were calculated as 0.02–8.33 ng/mL and 0.05–25.00 ng/mL. The recovery was determined to be 89.00–118.08% and precision was assessed with a coefficient of variation value of 1.74–8.64%. In the established LC–MS/MS MRM method, all markers in GHT samples were detected at 0.003–16.157 mg/g. Information gathered during the development and verification of the LC–MS/MS method will be useful for the quality assessment of GHT and other herbal medicines.


Introduction
Traditional herbal medicines, traditional Korean medicines (TKMs), traditional Chinese medicines (TCMs), and Kampo medicines (KMs), characterized by multiple components and multiple targets, have long been used in Asian countries, especially Korea, China, and Japan, for the treatment of and protection against various diseases, and for maintaining health [1]. These TKMs, TCMs, and KMs consist of combinations of at least two or more medicinal herbs and are taken in the form of decoction [2,3].
To date, many researchers have used analytical techniques such as HPLC, LC-MS/MS, and gas chromatography-mass spectrometry for the quality control of complex formulations such as TKMs, TCMs, and KMs [24][25][26][27][28]. Among the various analytical techniques, the analytical methods that include HPLC and LC-MS/MS are currently the most widely used for standardization purposes. In particular, the sensitive, accurate, and reliable LC-MS/MS system is being used in standardization studies for numerous phytochemical components that constitute TKMs, TCMs, and KMs [29].

Optimization of LC-MS/MS MRM Conditions
For the quality assessment of GHT using the marker analytes, we first determined the optimal simultaneous determination conditions in LC-MS/MS MRM mode. Consequently, the nine markers (liquiritin apioside, neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, liquiritigenin, glycyrrhizin, and 6-shogaol) were separated using gradient elution with a distilled water solution (containing 5 mM ammonium formate and 0.1% [v/v] formic acid)-acetonitrile mobile phase system on an Acquity UPLC BEH C 18 column (2.1 × 100 mm, 1.7 µm) maintained at 45 • C. Table 1 shows the optimal LC-MS/MS MRM parameters for the simultaneous quantification of each marker component. The established assay was successfully applied to the GHT sample and all markers were detected within 10 min, as shown in Figure 1 and Table 1.    Table 1). As MRM conditions for LC-MS/MS simultaneous analysis, the precursor ion (Q1) and product ion (Q3) peaks for each marker analyte were set as shown in Table 1. Liquiritin apioside, a flavanone, detected a Q3 ion peak at m/z 255.0 (M-H-Glc-Api] − ), generated by removal of the glucosyl-apisyl group [30].  [31][32][33]. Neoeriocitrin was detected at m/z 151.0 in the form of [ 1,3 A 0 -H] − , generated by retro-Diels-Alder (RDA) fragmentation of aglycone, from which rutinose had been removed [31,32]. The fragmentation of liquiritigenin is similar to that of neoeriocitrin; the Q3 ion peak was detected at m/z 137.0 ([M+H-4-vinylphenol] + ) by RDA cleavage [31]. Glycyrrhizin was in the form of [di-GlcA-H] − , in which aglycone was lost, and a Q3 ion peak was detected at m/z 351.2 [30]. In 6-shogaol, the Q3 ion peak was detected at m/z 137.1 in the form of [M+H-C 9 H 15 O] + , by cleavage of the C1-C2 bond by the ketone functional group of the alkyl chain [34,35]. MS fragmentation for the simultaneous determination of each marker as described above is shown in Figure S2.

Method Validation of the Developed Analytical Method
In this research, the newly developed LC-MS/MS MRM analytical method for the simultaneous determination of the nine marker analytes in GHT samples was validated by evaluating the linearity, limit of detection (LOD), limit of quantification (LOQ), recovery, and precision. Table 2, Table 3, Table 4 show the results for various validation parameters. Briefly, the coefficient of determination (r 2 ) value, which means the linearity of the calibration curve prepared in different concentration ranges for each marker analyte, was 0.9950-0.9968, showing good linearity, and LOD and LOQ values were estimated to be 0.02-8.33 ng/mL and 0.05-25.00 ng/mL, respectively ( Table 2). The recovery test, calculated from Equation (1), was conducted to evaluate the accuracy of the developed method; it was determined to be 89.00-118.08% (Table 3). The acceptance criteria for recovery test for validation of analysis of traditional herbal medicines such as TKMs, TCMs, and KMs are generally accepted with ±20%, so results of our study show that they are suitable [36,37]. In the precision verification, the repeatability of the retention time and peak area of the marker analytes was evaluated as the coefficient of variation (CV) values (calculated from Equation (2)); it was determined to be 0.08-0.52% and 3.04-9.64%, respectively (Table S2). In addition, the CV (%) values of intra-and interday precisions of the nine marker analytes were also determined to be <10.00% (Table 4). From the above various verification results, indications are that the LC-MS/MS MRM assay developed in this study is suitable and appropriate as an analytical method for the quality evaluation of GHT.

Simultaneous Determination of the Nine Marker Analytes in GHT Samples Using the Developed LC-MS/MS MRM Assay
Simultaneous determination of the nine marker analytes in GHT samples was conducted using the LC-MS/MS MRM assay developed and validated in this study. All marker analytes (liquiritin apioside, neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, liquiritigenin, glycyrrhizin, and 6-shogaol) were eluted, at 1.57, 1.58, 1.86, 1.99, 2.13, 2.27, 3.05, 4.95, and 8.50 min, respectively (Table 1 and Figure S3). The nine marker analytes in the lyophilized GHT samples were detected at 0.003-16.157 mg/g. The detailed content of each marker compound is given in Table 5. Among these markers, narirutin, narigin, hesperidin, and neohesperidin, derived from Citri Unshius Pericarpium, Citri Unshius Pericarpium Immaturus, Aurantii Fructus Immaturus, were present in large amounts. These results suggest the possibility of them being useful as basic data for the analysis of quality assessment of GHT.

Plant Materials
Nine raw herbal medicines constituting GHT were purchased from Kwangmyungdang Medicinal Herbs (KMH; Ulsan, Korea), a herbal medicine supplier for pharmaceuticals, in November 2017. All medicinal herbs were used after morphological verification by Dr. Seung-Yeol Oh, president of KMH. Detailed information on all raw herbs is shown in Table S1. Specimens of the nine raw herbal medicines (2017KE58-1 to 2017KE58-5) were deposited at the KM Science Research Division, Korea Institute of Oriental Medicine.

Chemicals and Reagents
The nine standard compounds used as markers for quality assessment of GHT in this study are shown in Figure S1. These compounds were provided by commercial suppliers and used for LC-MS/MS analysis: liquiritin apioside (C 26 Germany). Methanol and acetonitrile were LC-MS grade and supplied by ThermoFisher Scientific (San Jose, CA, USA). Purified water was used, specifically, produced through a Vivagen water purification system (EXL3 Analysis 16, Seongnam, Korea). Formic acid (≥99.5%) was supplied by Fujifilm Wako Pure Chemical Co., Ltd. (Osaka, Japan) and ammonium formate (99.0%) by Kanto Chemical Co., Inc. (Tokyo, Japan).

Preparation of GHT Water Sample
A sample of GHT in water was prepared following the same protocol as that in other previously reported methods for preparing a herbal prescription [38] (see Table S1). After mixing nine herbal medicines (Cnidii Rhizoma, Pinelliae Tuber, and Poria Sclerotium, each 881.52 g; Citri Unshius Pericarpium, Citri Unshius Pericarpium Immaturus, and Aurantii Fructus Immaturus, each 441.94 g; Atracylodis Rhizoma Alba and Glycyrrhizae Radix et Rhizoma, each 220.97 g; and Zingiberis Rhizoma Recens, 587.68 g), in a weight ratio (w/w), 50 L of distilled water was added, and extraction was performed under pressure (0.98 bar) at 100 • C for 2 h. The extract was subsequently filtered through a sieve (53 µm mesh) and then freeze dried (PVTFD100R, IlShinBioBase, Yangju, Korea) to afford a powder sample of about 1.0 kg (yield 20.1%).

Preparation of Samples and Standard Solutions for LC-MS/MS Quantification of the Nine Marker Analytes in GHT Samples
To analyze simultaneously the nine marker analytes in a GHT sample using LC-MS/MS, 70% methanol was added to approximately 50 mg of the lyophilized GHT sample to make up a volume of 50 mL. The mixed sample solution was continuously subjected to ultrasonic extraction for 5 min and vortexing for 1 min. Prior to analysis, the prepared sample solution was diluted 50-fold with 70% methanol and filtered through a hydrophobic polytetrafluoroethylene membrane filter (0.2 µm; Pall Life Sciences, Ann Arbor, MI, USA).
A standard solution for each marker analyte was prepared at a concentration of 100.0 µg/mL, using methanol, and then stored at 4 • C until analysis.

LC-MS/MS Instrumentation and Experimental Conditions for Simultaneous Determination of the Nine Marker Analytes in GHT Samples
Simultaneous determination of the nine marker analytes in GHT samples by LC-MS/MS was conducted using a previously reported protocol [39,40]. Briefly, LC-MS/MS analysis was performed using a Waters Acquity UPLC system (Milford, MA, USA) coupled with a Waters Xevo TQ-XS triple quadrupole MS system. Markers were separated on an Waters Acquity UPLC BEH C 18 column (2.1 × 100 mm, 1.7 µm), maintained at 45 • C, using gradient elution with a distilled water solution (containing 5 mM ammonium formate and 0.1% [v/v] formic acid)-acetonitrile mobile phase system. Detailed experimental parameters of ultra-performance liquid chromatography and MS for simultaneous determination are summarized in Table S3.
The accuracy verification of the newly developed LC-MS/MS method was performed through the recovery test. In other words, the recovery (%) was determined by adding known concentrations of each standard marker analyte (low, medium, and high) to the GHT sample as shown in Table 3, and calculated from Equation (1).
The precision (repeatability, intraday precision, and interday precision) of our newly developed analytical method was evaluated by calculating the CV value of each parameter. The repeatability was validated by calculating the CV value of retention time and peak area of each marker analyte, after six measurements, using a standard solution. Intraday precision and interday precision were assessed with CV values calculated after measurements for within day and 3 consecutive days on the three concentrations, respectively. The CV (%) value was calculated from Equation (2).

Statistical Analysis
Data were expressed as mean, SD, and CV (%) using Microsoft Excel 2019 software (Microsoft Co., Redmond, WA, USA).

Conclusions
In this study, for the first time, a sensitive, accurate, and reliable LC-MS/MS MRM assay for efficient quality assessment of GHT, a traditional herbal prescription, was developed using nine selected marker analytes. The developed assay was evaluated by examining the linearity, LOD, LOQ, accuracy, and precision. The established LC-MS/MS MRM assay is expected to be useful in not only the efficient quality control of GHT, but also in further studies on other TKMs, TCMs, and KMs.
Supplementary Materials: The following supporting information can be downloaded, Figure S1: Chemical structures of the nine marker components in GHT; Figure S2: MS fragmentation of each marker analyte; Figure S3: Extracted ion chromatograms of each reference standard (A) and GHT sample (B) measured by LC-MS/MS MRM mode; Table S1: Composition of prepared GHT; Table S2: Repeatability of the nine marker analytes in the LC-MS/MS MRM assay (n = 6); Table S3:  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data can be found in this paper.

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