Simultaneous Determination of Fourteen Marker Compounds in the Traditional Herbal Prescription, Geumgwesingihwan, Using Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry

Geumgwesingihwan (GSH) is a traditional herbal prescription composed of eight medicinal herbs: Rehmannia glutinosa (Gaertn.) DC., Dioscorea japonica Thunb., Cornus officinalis Siebold and Zucc., Poria cocos Wolf, Paeonia suffruticosa Andrews, Alisma plantago-aquatica subsp. orientale (Sam.) Sam., Achyranthes bidentate Blume, and Plantago asiatica L. This study developed and validated an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method in the multiple reaction monitoring (MRM) mode for simultaneous determination of 14 compounds (allantoin, gallic acid, 5-(hydroxymethyl)furfural, geniposidic acid, oxypaeoniflorin, loganin, geniposide, paeoniflorin, ecdysterone, verbascoside, cornuside, benzoylpaeoniflorin, paeonol, and alisol B acetate) in GSH. The chromatographic separation of all marker analytes was carried out on an Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm) using gradient elution of a mobile phase of distilled water–acetonitrile containing 0.1% acetic acid. The newly established UPLC–MS/MS MRM method was validated by evaluating the linearity, the limits of detection and quantification, recovery, and precision. All markers were detected at concentrations of 6.94–4126.28 mg/kg. In addition, the recovery was 76.65–119.49% and the relative standard deviation value of the precision was 0.19–9.91%. The newly developed and validated UPLC–MS/MS assay will provide useful information for quality assessment of GSH.


Introduction
Traditional Chinese medicine (TCM), traditional Korean medicine (TKM), and Kampo medicine (KM) prescriptions are a complex composed of two or more herbal medicines. They have been widely manufactured and administered in Asian countries such as China, Korea, and Japan for the treatment of various diseases or health maintenance and promotion because of the multitarget feature by multicomponents [1].
For the quality control of TCMs, TKMs, and KMs composed of several herbal medicines, it is by no means easy to analyze the numerous components contained in them at the same time. Nevertheless, many studies have been conducted to achieve standardization of

Selection of Marker Components for Simultaneous Analysis Using UPLC-MS/MS
We performed a profiling analysis using an UPLC-MS/MS multiple reaction monitoring (MRM) method on the following components to determine the marker analytes for quality control of GSH: 5-(hydroxymethyl)furfural from R. glutinosa; allantoin and dioscin from D. japonica; gallic acid, loganin, morroniside, sweroside, cornin, and cornuside from C. officinalis; pachymic acid and polyporenic acid C from P. cocos; paeonol, paeoniflorin, benzoic acid, oxypaeoniflorin, and benzoylpaeoniflorin from P. suffruticosa; alisol B and alisol B acetate from A. orientale; ecdysterone from A. bidentate; and geniposide, geniposidic acid, and verbascoside from P. asiatica [8][9][10][11][12][13][14][15][16][17][18]. We attempted to detect a total of 21 components selected from each constituent herbal medicine of GSH in the sample. Figure S1 in Supplementary Materials shows the UPLC-MS/MS MRM chromatograms of these marker candidates in the positive and negative ion modes, and only 14 components were detected in the GSH sample. Therefore, we selected these 14 detected components as marker analytes for quality control of GSH.

Optimization of UPLC Operation Conditions and UPLC-MS/MS MRM Parameters for Simultaneous Analysis
Compounds 1-14 were isolated and eluted from all markers within 20 min on an Acquity UPLC BEH C 18 reversed-phase column (2.1 mm × 100 mm, 1.7 µm; Waters, Milford, MA, USA) maintained at 40 • C and gradient elution of a mobile phase system of distilled water-acetonitrile (both containing 0.1% acetic acid) (Table S1). UPLC-MS/MS MRM conditions for simultaneous quantification of 14 marker analytes selected for the quality evaluation of GSH were explored. For quantification of the product ion (Q3) from the precursor ion (Q1) of each marker under the optimized MRM conditions of the markers, the MRM peak data were acquired for approximately 0.5 min at the retention time of each marker, and detailed parameters are presented in Table 1. Figure 1 shows the UPLC-MS/MS MRM chromatograms obtained in the positive and negative ion modes by applying optimized analysis conditions. The blank chromatogram of each analyte is shown in Figure S2.    (8,43.08 µg/L), ecdysterone (9, 6.43 µg/L), verbascoside (10, 3.83 µg/L), cornuside (11,4.00 µg/L), benzoylpaeoniflorin (12,8.00 µg/L), paeonol (13, 30.19 µg/L), and alisol B acetate (14, 1.50 µg/L).     [20]. The Q3 peaks for compounds 10, 13, and 14 were detected at m/z 163.0, 42.9, and 97.0 from which C 20 H 29 O 12 , C 7 H 7 O 2 , and C 26 H 40 O 4 were eliminated, respectively [26,27]. In compound 11, the Q3 peak was detected at m/z 211.0 ([M + H − Glu − trihydroxybenzoic acid] + ) from which glucose and trihydroxybenzoic acid groups were eliminated from the Q1 peak of m/z 543.1 in the form of [M + H] + [28]. The Q1/Q3 mass spectra of 14 marker components are presented in Figure S3.

Plant Materials
As shown in Table S3, eight medicinal herbs constituting GSH were purchased from Kwangmyungdang Pharmaceutical (Ulsan, Korea) in November 2017. Each raw medicinal herb was morphologically identified according to the guideline "The Dispensatory on the Visual and Organoleptic Examination of Herbal Medicine" by Dr. Goya Choi, Korea Institute of Oriental Medicine (KIOM, Daejeon, Korea) [29]. Specimens (2018CA04-1 to 2018CA04-8) of each raw material have been deposited at the KM Science Research Division, KIOM.

Preparation of GSH Water Extract
GSH water extract (GSH-1) was prepared in KIOM according to the sample preparation protocol of a previously reported study [30,31]. Briefly, after mixing the eight herbal medicines based on the weight presented in Table S3, 50 L of water was added and extracted at 100 • C for 2 h. To obtain a powder sample, the extract was freeze-dried (GSH-1: 1355.6 g; yield: 27.1%). The prepared sample was refrigerated until use. Another water extract of GSH (GSH-2) was provided by the College of Oriental Medicine, Wonkwang University.

Preparation of Sample Solutions and Standard Solutions for UPLC-MS/MS MRM Analysis
Sample solutions for UPLC-MS/MS MRM analysis of compounds 1-14 in GSH samples were prepared at concentrations of 142.0 mg/100 mL (GSH-1) and 133.0 mg/100 mL (GSH-2) using 50% methanol as solvent. Compound 3 was used for quantitation by diluting the prepared sample solution 10-fold.
These prepared solutions were filtered through a 0.22 µm membrane filter (Pall Life Sciences, Ann Arbor, MI, USA) before UPLC-MS/MS analysis.

UPLC-MS/MS MRM Analysis Conditions for Simultaneous Determination of the 14 Marker Analytes in GSH
UPLC-MS/MS MRM analysis for quantitative analysis of compounds 1-14 was applied to GSH samples by modifying the previously reported analysis protocol [31]. Briefly, a Waters Acquity UPLC H-Class (Milford, MA, USA) coupled with Xevo TQ-S micro MS system (Milford, MA, USA) was used, which was data accumulated and controlled using MassLynx (version 4.2; Milford, MA, USA). Compounds 1-14 were separated and quantified using an Acquity UPLC BEH C 18 column (2.1 mm × 100 mm, 1.7 µm; Milford, MA, USA) and gradient elution of a distilled water-acetonitrile mobile phase system both containing 0.1% (v/v) acetic acid. Detailed operating parameters of the equipment are shown in Table S1, and the MRM analysis conditions of each marker analyte for simultaneous analysis are summarized in Table 1.

Method Validation of the Established UPLC-MS/MS MRM Assay for Quality Control of GSH
Various factors such as the linearity, LOD, LOQ, recovery, and precision were investigated based on the guidelines to develop a simultaneous UPLC-MS/MS analysis method for compounds 1-14 in GSH samples and to validate the analysis method [32]. The linearity was validated by the r 2 value of the calibration curve prepared in the linearity range of each marker analyte shown in Table 2, and ≥0.99 was set as an appropriate range. At the same time, the LOD and LOQ values were calculated using the signal-to-noise ratio (S/N): LOD = 3 × S/N and LOQ = LOD × 3. Next, the recovery was evaluated using the standard addition method. That is, extraction and analysis were conducted by adding three different concentrations (low, medium, and high) of each known marker analyte to the GSH sample, and the recovery was calculated: recovery (%) = (found amount/spiked amount) × 100. The repeatability was measured six times using a standard solution, and then the CV value for the retention time of each marker analyte was evaluated. In addition, intra-and inter-day precisions were assessed using the CV values of compounds 1-14 measured for one day and three consecutive days, respectively.

Conclusions
In the present study, a highly accurate and sensitive UPLC-MS/MS system was firstly developed for simultaneous analysis of compounds 1-14 and to use it as basic data for quality control of a traditional herbal prescription, GSH. The newly UPLC-MS/MS MRM analytical method was developed satisfactorily, and the developed assay was validated by examining the linearity, LOD, LOQ, recovery, and precision. Furthermore, this method will be used as basic data for setting up a method for evaluating the quality of other TCMs, TKMs, and KMs prescriptions as well as GSH.