Isolation and Identification of Chemical Constituents from Zhideke Granules by Ultra-Performance Liquid Chromatography Coupled with Mass Spectrometry

Chemical constituents from Zhideke granules were rapidly isolated and identified by ultra-performance liquid chromatography (UPLC) coupled with hybrid quadrupole-orbitrap mass spectrometry (MS) in positive and negative ion modes using both full scan and two-stage threshold-triggered mass modes. The secondary fragment ion information of the target compound was selected and compared with the compound reported in databases and related literatures to further confirm the possible compounds. A total of 47 chemical constituents were identified from the ethyl acetate extract of Zhideke granules, including 21 flavonoids and glycosides, 9 organic acids, 4 volatile components, 3 nitrogen-containing compounds, and 10 other compounds according to the fragmentation patterns, relevant literature, and MS data. The result provides a new method for the analysis of chemical constituents of Zhideke granules which laid the foundation for quality control and the study of pharmacodynamic materials of Zhideke granules.


Introduction
Zhideke granules are an in-hospital preparation from Ruikang Hospital affiliated to the Guangxi University of Chinese Medicine. It contains 10 kinds of traditional Chinese medicines including Scutellaria baicalensis Georgi., Belamcanda chinensis (L.) Redouté, Mentha haplocalyx Briq., Eriobotrya japonica ( unb.), Platycodon grandiflorus (Jacq.) A. DC., Bupleurum chinense, Nepeta cataria L., Cynanchum glaucescens (Decne.) Hand-Mazz., Nervilia fordii (Hance) Schltr., and Sauropus spatulifolius Beille. e preparation that has been used in the treatment of bronchial asthma of wind-phlegm obstructing the lung in acute attack period for many years is widely used in Guangxi Zhuang Autonomous Region of China [1]. It has the efficacy of reducing fever and removing toxins, relieving cough and resolving phlegm. Moreover, it has been proven by longterm clinical practice that Zhideke granules have an effect on flu, fever, cough, bronchial asthma, etc. [2,3]. Although it has been reported that baicalin in Scutellaria baicalensis Georgi. and tectoridin and irisflorentin in Belamcanda chinensis (L.) Redouté are used as quality control components of Zhideke granules, the material base of it is still unclear. At the same time, some literatures about baicalin [4,5] and tectoridin [6,7] proved that they had antiasthma effect and reduced inflammation. However, other chemical constituents and pharmacodynamic substances of Zhideke granules have not been reported. e ultra-performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry (UPLC-Q-Orbitrap HRMS) is a new technology developed in recent years for analyzing the structure of complex traditional Chinese medicine (TCM) and its compound preparations [8][9][10][11], and it has the advantages of high efficiency, high speed, high sensitivity, and high resolution and specificity [12][13][14]. In this study, UPLC-Q-Orbitrap HRMS was used to rapidly isolate and identify unknown chemical components in Zhideke granules for the first time. We analysed the secondary fragment ion information of the target compound as well as relevant literature to further determine the possible chemical constituents in Zhideke granules, which has provided a reference for the quality control and its pharmacodynamics substances of Zhideke granules.

Chemicals and Reagents.
Zhideke granules were provided by Ruikang Hospital affiliated to Guangxi University of Chinese Medicine. UPLC-grade acetonitrile was purchased from Merck (Merck, Germany). UPLC-grade ammonium acetate was obtained from Shanghai Sixin Biotechnology Co., Ltd. (Shanghai, China). Formic acid and methanol at UPLC-grade were acquired from Fisher (Fisher, USA). Analytical grade ethyl acetate was purchased from Fisher (Fisher, USA). Ultra-pure water was purified with Milli-Q synergy (Millipore, USA). Forsythoside A (no. 111810-201606), baicalin (no. 110715-201720), and wogonin (no. 111514-201706) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Forsythoside A, baicalin, and wogonin purity were found to be above 97.2%, 93.5%, and 96.3%, respectively. Other chemicals were of analytical grade and their purity was above 99.5%.

Ethyl Acetate Extracts Preparation.
A 0.1 g sample of Zhideke granules was weighed by using a XS205DU electronic balance (Mettler-Toledo, Switzerland) and extracted with 20 mL of water in an ultrasonic bath (40 kHz, 500 W) for 30 min. en, the solutions were extracted twice with 20 mL ethyl acetate and combined two ethyl acetate extracts. Subsequently, these extracts were evaporated at the temperature of 80°C by water bath, and the residue was dissolved in 5 mL of methanol. Finally, the solution of the residue was filtered and was analysed.

2.
3. e Sample Solution of HPLC Analysis. A 2.0 g sample of Zhideke granule was precisely weighed and extracted with 10 mL of methanol in an ultrasonic bath for 30 min. en, the extract was centrifuged at 13000 r/min for 10 min by using a TGL-16G centrifuge (Shanghai Anting Scientific Instrument Factory, China) and the supernatant was taken as the sample solution. Finally, the solution was analysed by HPLC.

Standards Preparation.
We accurately weighed appropriate amounts of baicalin and wogonin, respectively, and then dissolved with methanol to prepare a mixture solution including two standards. Forsythoside A standard solution was prepared by dissolving 11.84 mg each of accurately weighed pure compound in 5 mL methanol.

Instruments and MS Conditions.
Chemical constituent's analyses were performed on ermo Fisher U3000 UPLC system ( ermo Fisher, USA), Trace Finder software ( ermo Fisher, USA), which was used for the UPLC-Q-Exactive Orbitrap MS data processing. e ion source was the heated electrospray ionization (ESI). e electrospray ionization source in both positive and negative ion modes was used in MS analysis. Spray voltages were set at 3.5 kV in a positive ion mode and 3.2 kV in a negative ion mode, respectively. e auxiliary gas temperature was 300°C, and the capillary temperature was 320°C. MS data were obtained on Full MS/dd-MS2 mode in the mass range of 100-1500 Da. e resolution of the precursor mass was 70000 FWHM, while the resolution of the product mass was 17500 FWHM. e specific ion scan mode was off. High purity nitrogen was used as the collision gas, and nitrogen was used as spray gas. e flow rates of sheath gas and auxiliary gas were at the rate of 30 and 10 μL/min, respectively. identified 47 possible chemical constituents including 21 flavonoids and glycosides, 9 organic acids, 4 volatile components, 3 nitrogen-containing compounds, and 10 other compounds from the ethyl acetate extract of Zhideke granules. e retention time, mass spectrometry information, and related literature of identified compounds are shown in Table 1.

Identification of Flavonoids and Glycosides.
ese compounds with a C 6 -C 3 -C 6 carbon skeleton in the structure called flavonoids had two benzene rings formed by three carbon atoms. e structure of flavonoids often results in substituents such as hydroxyl, methyl, and methoxyl groups. erefore, in the fragmentation regularity of flavonoids, these compounds easily lose neutral fragments of CO (28 Da), H 2 O (18 Da), CO 2 (44 Da), and fragment ions of substituents [15]. In addition, the retro-Diels-Alder (RDA) fragmentation is a common fragmentation pattern in flavonoids. Taking  [17] according to these results.
In nature, flavonoids mostly exist in the form of glycosides. Glycosidic bonds easily cleavage in the fragmentation pattern of glycosides. Taking  e possible fragmented pathway of compound 3 is shown in Figure 4 according to this fragmental information. us, compound 3 was characterized as puerarin [18].  Figure 5 according to this fragmental information. Compound 8 was baicalin by comparing with the reference standard and the literature report [15].

Azelaic acid
Cynanchum glaucescens (Decne.) Hand-Mazz [21] 6 Journal of Analytical Methods in Chemistry  [12] Note. a Identification confirmed with reference compound.      [21]. According to the fragmentation process [16], compound 29 was determined as abscisic acid. erefore, compound 30 was identified as salvianolic acid C by referring to the literature [22]. e fragmentation pathway of salvianolic acid C is shown in Figure 7. Based on fragment rules and literature [22], compound 28 was salvianolic acid A.

Identification of Other Compounds.
ere were some other compounds in the Zhideke granules, except constituents mentioned in the previous discussion such as sugar, coumarin, and phenol. Taking [29], compound 38 was identified as mannitol. e fragmentation pathway of mannitol is shown in Figure 9. Compound [36]. e fragmentation pathway of lobetyolin is shown in Figure 10.