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

Huang, Guangqiang; Liang, Jie; Chen, Xiaosi; Lin, Jing; Wei, Jinyu; Huang, Dongfang; Zhou, Yushan; Sun, Zhengyi; Zhao, Lichun

doi:https://doi.org/10.1155/2020/8889607

Journal of Analytical Methods in Chemistry

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Abstract Introduction Materials and Methods Results and Discussion Conclusions Data Availability Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2020 | Article ID 8889607 | https://doi.org/10.1155/2020/8889607

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

Guangqiang Huang,¹Jie Liang,^1,2,3Xiaosi Chen,¹Jing Lin,¹Jinyu Wei,¹Dongfang Huang,¹Yushan Zhou,¹Zhengyi Sun,⁴and Lichun Zhao^1,3

Academic Editor: Luca Campone

Received25 Aug 2020

Accepted08 Dec 2020

Published29 Dec 2020

Abstract

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.

1. 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 (Thunb.), Platycodon grandiflorus (Jacq.) A. DC., Bupleurum chinense, Nepeta cataria L., Cynanchum glaucescens (Decne.) Hand-Mazz., Nervilia fordii (Hance) Schltr., and Sauropus spatulifolius Beille. The 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 long-term 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.

The 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 [8–11], and it has the advantages of high efficiency, high speed, high sensitivity, and high resolution and specificity [12–14]. 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.

2. Materials and Methods

2.1. 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%.

2.2. 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. Then, 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. The 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. Then, 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.

2.4. 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.

2.5. HPLC Chromatographic Conditions

Separation was achieved on Agilent ZORBAX Eclipse Plus-C18 column (4.6 × 250 mm, 5 μm). The mobile phase was methanol (A) and 0.2% phosphoric acid (B) by gradient elution (0–30 min, 5%–20% A; 30–50 min, 20%–35% A; 50–75 min, 35%–40% A; 75–110 min, 40%–60% A; 110–150 min, 60%–90% A) with photo-diode array (PDA) detection at 254 nm at the flow rate of 0.8 mL/min. The column temperature was 30°C and the volume was 10 μL.

2.6. UPLC Chromatographic Conditions

In the UPLC system, the column was a Thermo Hypersil Gold C18 column (2.1 mm × 100 mm, 1.9 μm). The mobile phase consisted of 0.1% formic acid acetonitrile (A) and 0.1% formic acid water containing 10 mmoL of ammonium acetate (B) which was programmed with a gradient elution (0–2.0 min, 5% A; 2.0 to 42.0 min, 5% to 95% A; 42.0 to 47.0 min, 95% A; 47.1 to 50 min, 95% to 5% A) at a flow rate of 0.3 mL/min. The sample injection volume was 1 μL. The column temperature was maintained at 35°C.

2.7. Instruments and MS Conditions

Chemical constituent’s analyses were performed on Thermo Fisher U3000 UPLC system (Thermo Fisher, USA), Trace Finder software (Thermo Fisher, USA), which was used for the UPLC-Q-Exactive Orbitrap MS data processing. The ion source was the heated electrospray ionization (ESI). The 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. The 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. The resolution of the precursor mass was 70000 FWHM, while the resolution of the product mass was 17500 FWHM. The specific ion scan mode was off. High purity nitrogen was used as the collision gas, and nitrogen was used as spray gas. The flow rates of sheath gas and auxiliary gas were at the rate of 30 and 10 μL/min, respectively.

3. Results and Discussion

We chose 50% ethyl acetate as the extraction solvent and identified chemical constituents from the ethyl acetate extract of Zhideke granules by UPLC-Q-Orbitrap MS in this paper. In the positive and negative ion modes, the total ion chromatograms of ethyl acetate extract in Zhideke granules are shown in Figure 1. The HPLC spectra of the standard of forsythoside A (A), the sample solution (B), a mixture of two standards (C), and the HPLC fingerprint of Zhideke granules (D) are shown in Figure 2.

(a)

(b)

(a)

(b)

(c)

(d)

According to MS mass, MS/MS fragmentation information, fragmentation patterns, and literature reports, we 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. The retention time, mass spectrometry information, and related literature of identified compounds are shown in Table 1.

3.1. Identification of Flavonoids and Glycosides

These compounds with a C₆–C₃–C₆ carbon skeleton in the structure called flavonoids had two benzene rings formed by three carbon atoms. The structure of flavonoids often results in substituents such as hydroxyl, methyl, and methoxyl groups. Therefore, in the fragmentation regularity of flavonoids, these compounds easily lose neutral fragments of CO (28 Da), H₂O (18 Da), CO₂ (44 Da), and fragment ions of substituents [15]. In addition, the retro-Diels–Alder (RDA) fragmentation is a common fragmentation pattern in flavonoids. Taking compound 11 as an example, compound 11, with the quasimolecular ion m/z 301.0354 [M-H]⁻ and formula of C₁₅H₁₀O₇, was identified as quercetin in the negative ionization mode. The fragment ion at m/z 273.0396 [M-H-CO]⁻ was derived from the ion at the m/z 301.0354 by loss of a CO (28 Da) molecule in the MS/MS spectrum. The fragment ion at m/z 178.9978 [M-H-C₇H₆O₂]⁻ was from the ion at m/z 301.0354 because of the RDA fragmentation. Subsequently, fragment ions at m/z 151.0031 [M-H-C₇H₆O₂-CO]⁻ and m/z 107.0135 [M-H-C₇H₆O₂-CO-CO₂]⁻ originated from the ion at m/z 178.9978 by the loss of a CO (28 Da) molecule and a CO₂ (44 Da) molecule, respectively. Compound 11 was recognized as quercetin and the possible fragment pathway is as shown in Figure 3 according to these results [16]. Compound 17, with the quasimolecular ion m/z 283.0602 [M-H]⁻ and formula of C₁₆H₁₂O₅, was characterized as wogonin by comparing with the reference. Compound 18, with the quasimolecular ion m/z 403.1387 [M + H]⁺ and formula of C₁₅H₁₀O₇, was identified as nobiletin in the positive ionization mode. The ion at m/z 373.0900 [M + H-2CH₃]⁺ was from the ion at m/z 403.1387 by loss of two methyl group fragments. Subsequently, the ion at m/z 327.0849 [M + H-2CH₃-H₂O-CO]⁺ originated from the ion at m/z 373.0900 by loss of H₂O (18 Da) and CO (28 Da). Compound 18 was characterized as nobiletin [17] according to these results.

According to the fragmentation process of flavonoids, we presumed that compounds 9, 10, 12, 15, 16, 19, 20, and 21 were identified as scutellarein, eriodictyol, eupafolin, irigenin, baicalein, chrysin, pinocembrin, and tectorigenin, respectively.

In nature, flavonoids mostly exist in the form of glycosides. Glycosidic bonds easily cleavage in the fragmentation pattern of glycosides. Taking compound 3 as an example with the quasimolecular ion m/z 417.1159 [M + H]⁺ and formula of C₂₁H₂₀O₉, it was identified as puerarin in the positive ionization mode. The ion at m/z 399.1055 [M + H-H₂O]⁺ was due to a natural loss of a H₂O molecule (18 Da). Subsequently, the ion at m/z 297.07435 [M + H-H₂O-C₄H₆O₃]⁺ originated from the ion at m/z 373.0899 by the loss of C₄H₆O₃ (102 Da) fragment. Moreover, the fragment ion at 351.0837 [M + H-2H₂O-CH₂O]⁺ came from the ion at m/z 399.1055 by the loss of a H₂O molecule (18 Da) and CH₂O fragment ion (39 Da). Fragment ions at m/z 267.0640 [M + H-2H₂O-CH₂O-C₄H₄O₂]⁺ and m/z 307.0941 [M + H-2H₂O-C₂H₄O]⁺ were derived from the fragment ion m/z 351.0837 by the loss of a C₄H₄O₂ (84 Da) fragment and a C₂H₄O (44 Da) fragment, respectively. The possible fragmented pathway of compound 3 is shown in Figure 4 according to this fragmental information. Thus, compound 3 was characterized as puerarin [18].

Compound 8, with the quasimolecular ion m/z 447.0902 [M + H]⁺ and formula of C₂₁H₁₈O₁₁, was identified as baicalin in the positive ion mode. The ion at m/z 447.0902 produced the ion at m/z 271.0588 [M + H-C₆H₈O₆]⁺ after the loss of a glucuronic acid molecule (177 Da). The fragmented ion m/z 169.0124 [M + H-C₆H₈O₆-C₈H₆]⁺ was from the ion at m/z 271.0588 by the loss of a C₈H₆ fragment ion (102 Da). Subsequently, the ion at m/z 225.054 [M + H-C₆H₈O₆-C₈H₆-H₂O-CO]⁺ originates from the ion at m/z 271.0588 by loss of a H₂O molecule (18 Da) and a CO molecule (28 Da). The possible fragment pathway of compound 8 is shown in Figure 5 according to this fragmental information. Compound 8 was baicalin by comparing with the reference standard and the literature report [15].

Based on the fragmentation patterns of glycosides, compounds 1, 2, 4, 5, 6, 7, 13, 14, and 20 were possibly identified as isoquercitrin, tectoridin, diosmin, hesperidin, luteolin 7-glucuronide, iridin, linarin, didymin, and pnocembrin, respectively.

3.2. Identification of Organic Acids

Organic acids of Zhideke granules are mainly found in Eriobotrya japonica (Thunb.), Platycodon grandiflorus (Jacq.) A. DC., Mentha haplocalyx Briq., etc. Organic acids mainly include two types, one of which is fatty acid and the other is aromatic acid. Organic acids easily lose neutral fragments of CO (28 Da), H₂O (18 Da), CO₂ (44 Da), and a fragment of alkyl. Taking compound 24 as an example, compound 24, with the quasimolecular ion m/z 197.0448 [M-H]⁻ and formula of C₉H₁₀O₅, was identified as danshensu under the negative ion mode. The fragment ion was at m/z 179.0342 [M-H-H₂O]⁻ at the loss of a H₂O molecule. Fragment ions at m/z 151.0401 [M-H-H₂O-CO]⁻ and m/z 134.0369 [M-H-H₂O-CO₂]⁻ were derived from the fragment ion m/z 179.0343 by the loss of a CO (28 Da) molecule and a CO₂ (44 Da) molecule, respectively. Based on fragment rules and literature, compound 24 was characterized as danshensu [19].

Compound 26, with the quasimolecular ion at m/z 537.1024 [M-H]⁻ and formula of C₂₇H₂₂O₁₂, was identified as lithospermic acid under the negative ion mode. The fragment ion at m/z 493.1024 [M-H]⁻ was formed due to the loss of a CO₂ (28 Da) molecule of the ion at m/z 537.1024. Fragment ions at m/z 295.0602 [M-H-CO₂-C₉H₁₀O₅]⁻ and m/z 313.0711 [M-H-C₉H₈O₄]⁻ were derived from the ion at m/z 493.1027 by the loss of fragment ions of C₉H₁₀O₅ (198 Da) and C₉H₈O₄ (180 Da). According to the results, compound 26 was identified as lithospermic acid [20]. The fragmentation pathway is shown in Figure 6.

Compound 27, with the quasimolecular ion at m/z 187.0968 [M-H]⁻ and formula of C₉H₁₆O₄, was identified as azelaic acid under the negative ion mode. The fragment ion at m/z 143.1070 [M-H-CO₂]⁻ was formed due to the loss of a CO₂ (28 Da) molecule of the ion at m/z 187.0968. The fragment ion at m/z 125.0967 [M-H-CO₂-H₂O]⁻ was derived from the ion at m/z 143.1070 by the loss of an H₂O (18 Da) molecule. Subsequently, the fragment ions at m/z 97.0654 [M-H-CO₂-H₂O-C₂H₄]⁻, m/z 83.0501 [M-H-CO₂-H₂O-C₃H₆]⁻, and m/z 69.0342 [M-H-CO₂-H₂O-C₄H₈]⁻ were derived from the ion at m/z 125.0967 by the loss of fragment ions of C₂H₄ (28 Da), C₃H₆ (42 Da), and C₄H₈ (56), respectively. Based on MS data and related literature, compound 27 was identified as azelaic acid [21]. According to the fragmentation process [16], compound 29 was determined as abscisic acid.

Compound 30, with the quasimolecular ion at m/z 491.0971[M-H]⁻ and formula of C₂₆H₂₀O₁₀, was identified as salvianolic acid C under the negative ion mode. The fragment ions at m/z 135.0446 [M-H-C₁₈H₁₂O₈]⁻, 179.0342[M-H-C₁₇H₁₂O₆]⁻, m/z 197.0971 [M-H-C₁₇H₁₀O₅]⁻, and m/z 311.0554 [M-H-C₉H₈O₄]⁻ were from the ion at m/z 491.0970 by the loss of fragment ions C₁₈H₁₂O₈ (356 Da), C₁₇H₁₂O₆ (312 Da), C₁₇H₁₀O₅ (294 Da), and C₉H₈O₄ (180 Da), respectively. Subsequently, the fragment ion at m/z 265.0501 [M-H-C₉H₈O₄-COOH]⁻ was made up of the ion at m/z 311.0554 by the loss of fragment ions of the carboxyl group fragment. The ion at m/z 293.0448 [M-H-C₉H₁₀O₅]⁻ was from the cleavage of the ion at m/z 491.0970 occurring in the position of the C-O bond in the ester bond. Therefore, compound 30 was identified as salvianolic acid C by referring to the literature [22]. The fragmentation pathway of salvianolic acid C is shown in Figure 7. Based on fragment rules and literature [22], compound 28 was salvianolic acid A.

Based on the fragmentation rules of organic acids, compounds 22, 23, and 25 were identified as gallic acid, pyrogallol, and p-hydroxycinnamic acid, respectively.

3.3. Identification of Volatile Components

Volatile components are mainly found in Belamcanda chinensis (L.) Redouté, Cynanchum glaucescens (Decne.) Hand.-Mazz., and Bupleurum chinense in Zhideke granules. Taking compound 31 as an example, compound 31, with the quasimolecular ion at m/z 127.0385 [M + H]⁺ and formula of C₆H₆O₃, was identified as 5-hydroxymethylfurfural in the positive ion mode. The fragment ion at m/z 127.0385 lost the CH₂OH group (31 Da) and produced the fragment ion at m/z 97.0285 [M + H-CH₂OH]⁺. The fragment ion at m/z 81.0337[M + H-H₂O-CO]⁺ was derived from the ion at m/z 127.0385 by the loss of a H₂O (18 Da) molecule and a CO (28 Da) molecule, successively. Then, the fragment ion at m/z 81.0337 continuously lost a CO (28 Da) molecule and yielded the fragment ion at m/z 53.0392 [M + H-H₂O-2CO]⁺. Based on MS data and relevant literature [23], compound 31 was identified as 5-hydroxymethylfurfural.

Compound 32 showed m/z 153.1267 [M + H]⁺ ion and a formula of C₁₀H₁₆O in the positive ion mode of the first-order mass spectrum. In the MS/MS spectrum, we observed fragment ions at m/z 59.0496, 69.0701, 93.0699, 95.0854, 97.0646, 107.0854, 109.1008, and 135.1165. These results of fragmentation patterns are consistent with the relevant literature [24]. Thus, compound 32 was identified as camphor.

Compound 33, with the quasimolecular ion at m/z 191.1058 [M + H]⁺ and formula of C₁₂H₁₄O, was identified as ligustilide in the positive ion mode. Fragment ions at m/z 163.1110 [M + H-C₂H₄]⁺ and m/z 173.0953 [M + H-H₂O]⁺ were from the ion at m/z 191.1058 by the loss of C₂H₄ (28 Da) fragment and an H₂O (18 Da) molecule, respectively. Subsequently, the fragment ion at m/z 173.0953 [M + H-H₂O]⁺ was derived from the ion at m/z 173.0953 by the loss of a CO (28 Da) molecule. According to relevant literature [25], compound 33 was identified as ligustilide. The fragmentation pathway of compound 33 is given in Figure 8.

Compound 34, with the quasimolecular ion at m/z 315.2528 [M + HCO₂]⁻ and formula of C₁₇H₃₄O₂, was identified as methyl palmitate in the positive ion mode. Fragment ions at m/z 127.1122 C₉H₁₉⁺, m/z 141.1279 C₁₀H₂₁⁺, were formed because of cleavage of the alkyl group of the ester compounds. In addition, the fragment ion at m/z 171.1020 [M-H-C₇H₁₅]⁻ was derived from the [M-H]⁻ ion. Thus, compound 34 was identified as methyl palmitate based on the fragmentation rules and related reports [23].

3.4. Identification of Nitrogen-Containing Compounds

Nitrogen-containing compounds refer to a class of organic compounds containing a nitrogen element in the structure of the molecule and mainly include a nucleoside, an amino acid, and nicotinamide. The nitrogen-containing compounds are mainly derived from Mentha haplocalyx Briq. and Bupleurum chinense in Zhideke granules. The natural loss of H₂O (18 Da), CO₂ (44 Da), and NH₃ (17 Da) molecules easily takes place in the fragmentation process of nitrogen-containing compounds. Besides, amino acid molecules easily lost the carboxyl group (45 Da) and a hydroxyl group (17 Da).

Compound 35, with the quasimolecule ion at m/z 195.0867 [M + H]⁺ and formula of C₈H₁₀N₄O₂, was identified as caffeine in the positive ion mode. The fragment ions at m/z 138.0656 [M + H-C₂H₄N₂]⁺, m/z 110.0711 [M + H-C₄H₁₀N₂]⁺, m/z 69.0451 [M + H-C₅H₁₀N₃O]⁺, and m/z 56.0500 [M + H-C₆H₆N₂O₂]⁺ were derived from the fragment ion at m/z 195.0867 by the loss of the fragment ions of C₂H₄N₂ (56 Da), C₄H₁₀N₂ (86 Da), C₅H₁₀N₃O (128 Da), and C₆H₆N₂O₂ (138 Da), respectively. The fragmentation patterns are basically consistent with the literature [26]. Thus, compound 35 was identified as caffeine. According to the fragmentation pattern, compound 36 was identified as adenine [27].

Compound 37, with the quasimolecule ion at m/z 123.0548[M + H]⁺ and formula of C₆H₆N₂O, was identified as nicotinamide in the positive ion mode. The fragment ions at 105.0445 [M + H-NH₃]⁺ and m/z 95.0606 [M + H-CO]⁺ were from the ion at m/z 123.0548 by the loss of a NH₃ (17 Da) molecule fragment and a CO (28 Da) molecule, respectively. Besides, the fragment ion at m/z 80.0497 [M + H-CO-NH₂]⁺ was formed owing to the loss of the NH₂ (16 Da) fragment. Therefore, compound 37 was identified as nicotinamide [28].

3.5. Identification of Other Compounds

There were some other compounds in the Zhideke granules, except constituents mentioned in the previous discussion such as sugar, coumarin, and phenol. Taking 38 as an example, compound 38, with the quasimolecular ion at m/z 181.0711 [M + H]⁺ and a formula of C₆H₁₄O₆, was identified as mannitol in the positive ion mode. The fragment ion at m/z 163.0670 [M-H-H₂O]⁻ was due to the loss of a H₂O (18 Da) molecule of the ion at m/z 181.0711. The fragment ions at m/z 101.0240 [M-H-H₂O-C₂H₆O₂]⁻, m/z 59.0136 [M-H-H₂O-C₄H₈O₃]⁻, and m/z89.0241 [M-H-H₂O-C₃H₆O₂]⁻ were formed from the fragment ion at m/z 163.0670 by the loss of the fragment ions of C₂H₆O₂ (62 Da), C₄H₈O₃ (104 Da), and C₃H₆O₂ (74 Da), respectively. Subsequently, the fragment ion at m/z 89.0241 [M-H-H₂O-C₃H₆O₂]⁻ lost a H₂O (18 Da) molecule and yielded m/z 71.0135 [M-H-2H₂O-C₃H₆O₂]⁻. Taking the literature into account [29], compound 38 was identified as mannitol. The fragmentation pathway of mannitol is shown in Figure 9.

Compound 39 showed m/z 341.1078 [M-H]⁻ ion and a formula of C₁₂H₂₂O₁₁ in the negative ion mode of the first-order mass spectrum. In the MS/MS spectrum, the fragment ions at m/z 59.0136, 71.0136, 89.0241, 95.0137, and 113.0241 were obtained. These fragmentation patterns were consistent with the literature report [25]. Therefore, compound 39 was recognized as sucrose.

Compound 40, with the quasimolecular ion at m/z 137.0238 [M-H]⁻ and formula of C₇H₆O₃, was identified as protocatechualdehyde in the negative ion mode. The characteristic fragment ion at m/z 93.0341 [M-H-CO₂]⁻ originates from the ion at m/z 137.0238 by the loss of a CO₂ (44 Da) molecule. Based on the reported literature [30], compound 40 was identified as protocatechualdehyde.

Compound 41, with the quasimolecular ion at m/z 177.0186 [M-H]⁻ and formula of C₉H₆O₄, was identified as esculetin in the negative ion mode. Fragment ions at m/z 133.0290 [M-H-CO2]⁻ and m/z 149.0237 [M-H-CO]⁻ were derived from the fragment ion at m/z 177.0186 by the loss of a CO₂ (44 Da) molecule and a CO (28 Da) molecule, respectively. Thus, compound 41 was identified as esculetin [31]. Meanwhile, compound 45 was identified as 5,7-dihydroxy-4-methyl coumarin according to the fragmentation pattern [32].

Compound 42, with the quasimolecular ion at m/z 421.0766 [M-H]⁻ and formula of C₁₉H₁₈O₁₁, was identified as isomangiferin in the negative ion mode. The fragment ion at 258.0161 [M-H-C₆H₁₀O₅]⁻ was formed from the cleavage of the ion at m/z 421.0766 occurring in the position of the glucoside bond by the loss of the C₆H₁₀O₅ (162 Da) fragment ion. Therefore, compound 42 was identified as isomangiferin [33].

Compound 43, with the quasimolecular ion at m/z 243.0655 [M-H]⁻ and formula of C₁₄H₁₂O₄, was identified as piceatannol in the negative ion mode. The fragment ions at m/z 225.0549 [M-H-H₂O]⁻, m/z 159.0445 [M-H-C₄H₄O₂], and m/z 201.0549 [M-H-C₂H₂O]⁻ originated from the ion at m/z 243.0655 by the loss of a H₂O (18 Da) molecule, a C₄H₄O₂ (84 Da) fragment, and a C₂H₂O (42 Da) fragment. Subsequently, the fragment ion m/z 173.0601 [M-H-C₂H₂O-CO]⁻ was formed from the ion at m/z 201.0549 [M-H-C₂H₂O]⁻ due to a CO (28 Da) molecule. Compound 43 was identified as piceatannol [34].

Compound 44 showed m/z 623.1963[M-H]⁻ ion and a formula of C₂₉H₃₆O₁₅ in the negative ion mode of the first-order mass spectrum. Fragment ions at m/z 461.1656 [M-H-C₉H₆O₃]⁻ and m/z 179.0341 [M-H-C₂₀H₂₈O₁₁]⁻ were derived from the fragment ion at m/z 623.1966 by the loss of the fragments C₉H₆O₃ (162 Da) and C₂₀H₂₈O₁₁ (444 Da), respectively. Furthermore, the fragment ion at m/z 179.0341 lost a H₂O (18 Da) molecule and yielded the fragment ion at m/z 161.0238 [M-H-C₂₀H₂₈O₁₁-H₂O]⁻. Based on relevant literature [35], compound 44 may be forsythoside A, isoacteoside, or verbascoside. Then, compound 44 was identified as forsythoside A by comparing with the reference standard.

Compound 46, with the quasimolecular ion at m/z 441.1754 [M + HCOOH]⁻ and formula of C₂₀H₂₈O₈, was identified as lobetyolin in the negative ion mode. The fragment ion at m/z 185.0963 [M-H-C₆H₁₀O₆-CH₂O]⁻ was derived from the ion at m/z 441.1754 by the loss of a fragment of the C₆H₁₀O₆ (178 Da) and CH₂O (30 Da) groups. Subsequently, the fragment ion at m/z 159.0811 [M-H-C₆H₁₀O₆-CH₂O-C₃H₆O]⁻ originated from the ion at m/z 185.0963 by the loss of C₃H₆O (58 Da) fragment. In addition, the fragment ion at m/z 89.0241 [M-C₁₃H₂₂O₈]⁻ was formed from the ion at m/z 441.1754 by the loss of C₁₃H₂₂O₈ fragment. Thus, compound 46 was identified as lobetyolin [36]. The fragmentation pathway of lobetyolin is shown in Figure 10.

Compound 47, with the quasimolecular ion at m/z 209.0799 [M + H]⁺ and formula of C₁₁H₁₂O₄, was identified as methyl 4-hydroxy-3-methoxycinnamate in the positive ion mode. Fragment ions at m/z 177.0540 [M-H-CH₄O]⁺ and m/z 149.0492 [M-H-C₂H₄O₂]⁺ were derived from the ion at m/z 209.0799 by the loss of fragments of CH₄O (32 Da) and C₂H₄O₂ (60 Da). Subsequently, the fragment ion at m/z 134.0593 [M-H-C₂H₄O-CH₃]⁺ was formed from the ion at m/z 149.0492 by loss of the CH₃ (15 Da) fragment. Therefore, compound 47 was identified as ferulic acid methyl ester [12]. The fragmentation pathway of ferulic acid methyl ester is shown in Figure 11.

4. Conclusions

The paper provided a new analysis method for the chemical constituents research of Zhideke granules. At the same time, this study rapidly and systematically analysed the chemical constituents of Zhideke granules by UPLC coupled with hybrid quadrupole-orbitrap MS. 47 chemical constituents were identified by UPLC-Q-Orbitrap HRMS including flavonoids and glycosides, organic acids, volatile components, nitrogen-containing compounds, sugars, and coumarins, which will provide a reference for quality control of Zhideke granules, and further reveal the pharmacodynamic substances of Zhideke granules.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Acknowledgments

Financial support was provided by Special Fund Project for Innovation Driven Development of Guangxi (AA17202046), The First-Class Subject of Traditional Chinese Medicine (Ethnic Pharmacy) in Guangxi ([2018]12), Collaborative Innovation Center of Zhuang and Yao Medicine ([2013]20), Guangxi Province Key Laboratory of Zhuang and Yao Medicine ([2014]32), The Eighth Batch of Special Experts Project of Guangxi Zhuang Autonomous Region (Study on quality standard of Zhuang and Yao medicine, [2019]13), Cultivating High-Level Talent Teams in the Qi Huang Project of Guangxi University of Chinese Medicine (2018002), Program for Innovative Research Team of High Education and Outstanding Scholar in Guang Xi ([2019]52), and Guangxi One Thousand Young and Middle-Aged College and University Backbones Teachers Cultivation Program ([2019]5).

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Copyright © 2020 Guangqiang Huang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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