Qualitative Analysis of Multiple Phytochemical Compounds in Tojapride Based on UHPLC Q-Exactive Orbitrap Mass Spectrometry

Tojapride is composed of Caulis Perillae, Rhizoma Cyperi, Radix Glycyrrhizae, Citrus aurantium L., Coptis chinensis Franch, Pericarpium Citri Reticulatae, Reynoutria japonica Houtt, Tetradium ruticarpum, and Cleistocactus sepium. It has the effects of inhibiting gastric acid and relieving pain. It is clinically used for treating gastroesophageal reflux disease. To further study the pharmacodynamic properties of Tojapride, the systematic characterization of the chemical constituents in Tojapride was investigated using ultra-performance liquid chromatography with Q-Exactive Orbitrap mass spectrometry combined with parallel reaction monitoring for the first time. Eventually, a total of 222 compounds, including flavonoids, alkaloids, and glycyrrhizic acid derivatives, were identified based on the chromatographic retention times, MS/MS2 information, and bibliography data; a total of 218 of these were reported for the first time as being present in Tojapride. This newly developed approach provides a powerful tool for extending our understanding of chemical constituents of Tojapride, which can be further extended to other TCMP composition research.


Introduction
Tojapride is a Chinese medicine preparation composed of Caulis Perillae, Rhizoma Cyperi, Radix Glycyrrhizae, Citrus aurantium L., Coptis chinensis Franch, Pericarpium Citri Reticulatae, Reynoutria japonica Houtt, Tetradium ruticarpum, and Cleistocactus sepium. Tojapride is used for improving symptoms such as heartburn, chest pain, acid reflux, and nausea, and it is mainly used for treating gastroesophageal reflux disease (GERD), which is one of the most common chronic, progressive upper gastrointestinal tract disorders of the esophagus, characterized by heartburn and regurgitation symptoms [1,2]. It is reported that all of the single components in Tojapride have good pharmacological activities, including antibacterial, anti-inflammatory, and anti-tumor activities, and its use plays an important role in the treatment of GERD. However, according to the existing literature, there are few reports on the chemical composition of Tojapride. Thus, it is necessary to establish a valid method for systematically identifying the constituents of Tojapride, which will be beneficial to gain a deeper understanding of its pharmacodynamic properties of Tojapride.
Traditional Chinese medicine preparation (TCMP) has a long history in medical practice and health care, particularly in Asian and African countries. TCMP has attracted considerable attention in great many fields due to its effective therapeutic performance and low toxicity [3,4]. With the development of analytical techniques, the combination of ultra-high-performance liquid chromatography and mass spectrometry (UHPLC/MS) has had a significant impact on TCMP development in recent years [5,6]. They feature powerful analytical figures of merit (sensitivity, selectivity, speed of analysis) and have a wide scope of application in the qualitative and quantitative analysis of the TCMP [7].
In this research, an effective method using UHPLC with Q-Exactive Orbitrap mass spectrometry was established to characterize the chemical constituents of Tojapride. A total of 222 compounds were identified based on MS/MS 2 data confirmation, retention time, and structural speculation; a total of 218 compounds were reported for the first time as being present in Tojapride. The results lay a foundation for the quality control of this medicine in the clinical use of Tojapride in the future. Additionally, LC-MS/MS has been proven to be an effective method for identifying TCMPs, and it also provides a new platform for qualitative analysis in many fields.

Analytical Strategy
The purpose of this study was to systematically identify the chemical constituents of Tojapride. Hence, an ideal strategy based on UHPLC-Q-Exactive Orbitrap MS combined with parallel reaction monitoring (PRM) was established. Firstly, the compound in Tojapride was extracted and enriched by ultrasonic extraction with 70% methanol. Secondly, the sample was injected into an UHPLC-Q-Exactive Orbitrap Mass Spectrometer to gain highresolution MS data for Tojapride. Thirdly, the MS 2 data for the trace ingredients in Tojapride were collected by UHPLC-Q-Exactive Orbitrap MS combined with PRM scanning. Lastly, the additional candidate chemicals were identified based on comparison with standards, summary DFIS, and neutral loss, as well as through comparison with the literature.

Profiling of the Chemical Composition of Tojapride by LC-MS/MS
In total, 222 compounds were identified in Tojapride, including 133 flavonoids, and 39 glycyrrhizic acid derivatives, 22 alkaloids, and 28 others. Among them, 40 compounds were accurately identified by the reference standard. LC-MS/MS data regarding the chemical compound of Tojapride according to the LC-MS/MS analysis are presented in Table S1 (Supplementary Material). The high-resolution extracted ion flow diagram of Tojapride is shown in Figure 1  .0480 indicated that they possessed a liquiritigenin moiety. Thus, they were identified as being liquiritin apioside isomers [12]. Likewise, compounds 105 and 145 were deduced as being 6'-acetylliquiritin isomers [13].
Compounds 150, 161, 164, 182 [19]. The main product ions at m/z 404.1089 and 389.0863 were attributed to the successive loss of a CH 3 radical (15 Da), and the obtained product ions at m/z 371.0762 were attributed to the loss of H 2 O from the product ions at m/z 389.0863.
Compounds 189 and 194 were eluted at 29.15 and 29.73 min, respectively, possessing the same quasi-molecular ions [M+H] + at m/z 343.1176. The main daughter ion at m/z 313.0701 was attributed to the loss of a 2CH 3 radical (30 Da). The fragment ions were obtained at m/z 285.0754 due to the loss of a CO radical (28 Da), and they were deduced as being 5,7,3',4'-Tetramethoxyflavone isomers [19].

Characterization of the Alkaloids in Tojapride
Compound 31 was detected at 11.08 min, possessing the quasi-molecular ions [M+H] + at m/z 505.2306 and MS/MS fragment ion at m/z 342.1698, due to the neutral loss of glucose reside (162 Da), and this indicated the presence of magnoflorine. Thus, it was identified as magnoflorine-O-glucoside [5].
Compound 21 was eluted at 9.54 min, possessing the quasi-molecular ions [M+H] + at m/z 342.1705, and was identified as magnoflorine by comparing the retention times and MS and MS 2 information with those of the standards. Compounds 38, 49, and 57, which possessed the same quasi-molecular ions and characteristic fragment ions as magnoflorine, were characterized as being magnoflorine isomers.
Compounds 74, 82, and 90, detected at 14.30, 15.27, and 15.85 min, respectively, possessed the same quasi-molecular ions [M+H] + at m/z 324.1236 and the main fragment ion at m/z 309.0993 and 294.0759, generated by the sequential loss of the CH 3 group (15 Da); they were identified as demethyleneberberine [20].
Compounds 77, 98, and 116 had the quasi-molecular ions [M+H] + at m/z 322.1079, and yielded the fragment ions at m/z 307.0837 due to the neutral loss of a CH 3 (15 Da). The product ions at m/z 279.0887 were obtained by the loss of CO (28 Da) moieties from the main ion at m/z 307.0837. Hence, they were deduced as being groenlandicine [20].
Compounds 83, 102, 106, and 114 were eluted at 15.27, 16.93, 17.22, and 18.40 min, respectively, possessing the same quasi-molecular ions [M+H] + at m/z 338.1392, and were deduced as being columbamine [20]. The main daughter ion at m/z 322.1072 and 308.0915 was due to loss of a OH radical (16 Da)  acterized as palmatine [20] and had an MS/MS fragment ion at m/z 336.1226 and 308.1277, attributing to the loss of an OH radical (16 Da) and 2CH 3 radical (30 Da), respectively.
Compounds 100 and 108 were eluted at 16.90 and 17.53 min, respectively, and they yielded the parent ion [M+H] + at m/z 320.0923; they were deduced as being coptisine, according to the MS and MS/MS spectra [20].
Compounds 104, 121, and 129 were eluted at 17. 16,19.47, and 20.37 min, respectively, having the same precursor ion [M+H] + at m/z 336.1240, and the fragment ion at m/z 321.0994 and 320.0914, generated by the loss of the CH 3 residue (15 Da) and CH 3 -H residue (16 Da), respectively. This further gave rise to the product ions at m/z 292.0964 [M-CH 3 -H-CO] + , these compounds were tentatively proposed as being berberine isomers [20].

Characterization of the Glycyrrhizic Acid Derivative in Tojapride
Compounds 34 and 158, which appeared at a retention time of 11 . Hence, they were identified as being 22-hydroxyl-licorice saponin G2 isomers [21].

Other Chemical Constituents in
Compounds 3 and 7 were eluted at 1.29 and 2.05 min, respectively, with the quasimolecular ion [M-H] − at m/z 331.0671 and the fragmentation ion at m/z 169.0134, corre-sponding to the loss of one glucose moiety (162 Da). The product ions at m/z 169.0133 and 125.0232 indicated the presence of gallic acid. Therefore, compounds 3 and 7 were determined to be a 1-galloyl-β-glucose isomer [4].  [23]. Likewise, compounds 170 and 197 were tentatively identified as being glycycoumarin isomers, and compound 215 was a glycyrol isomer [11].
Compounds 10 and 12 were eluted at 4.03 and 5.57 min, and they possessed a similar molecular ion [M-H] − at m/z 153.0193 and fragment ion at m/z 109.0283 to protocatechuic acid. Accordingly, compound 135 was identified as a protocatechuic acid isomer. Likewise, compounds 13, 16, and 19 were identified as being an isoferulic acid isomers; compounds 45, 70, and 136 were characterized as being abscisic acid isomers.

Pharmacological Activity of Chemical Ingredients in the Tojapride
The compounds identified in this study included flavonoids, alkaloids, glycyrrhizic acid derivative, and other compounds. Flavonoids are widely distributed in various medicinal plants and are the effective components of many traditional Chinese medicines. According to reports, flavonoids have been repeatedly studied because of their favorable pharmacological activity. For example, as emodin, naringin, and neohesperidin can be used in the treatment of GI motility disorders, they may play an important role in the pharmacodynamic properties of Tojapride, which itself plays a role in pain relief, reduction in acid regurgitation, and the promotion of GI motility [24,25]. The anti-inflammatory activity of Tojapride was confirmed many years ago, and it has been widely used in patients with non-erosive reflux disease. Most of the compounds identified in this study, such as quercetin, magnoflorine, and glycyrrhizic acid, have anti-inflammatory activity, which may be the material basis for the treatment of GERD.

Materials and Chemicals
The chemical reagents (HPLC-grade), including acetonitrile and methanol, were purchased from Fisher Scientific (New Jersey, USA). The LC-MS-grade formic acid was acquired from Thermo Fisher Scientific Co., Ltd. (Carlsbad, CA, USA). The deionized water was prepared using a Milli-Q system (Bedford, MA, USA). Other solvents were of an analytical grade. Detailed information regarding the 40 standards is listed in Table S2. Tojapride was provided by the Xiyuan Hospital, China Academy of Chinese Medical Sciences (Beijing, China).

Preparation of Standard and Sample Solutions
A Tojapride sample (1 mg) was dissolved in 70% methanol/water (10 mL) in a 15 mL tube and vortexed (5 min). Subsequently, the sample was transferred into the ultrasonic machine for 0.5 h. Then, the solution was centrifuged at 15,000 RCF for 15 min and the supernatant was injected into an HPLC vial. The sample solution (3 µL) was then injected into the LC-MS system for further analysis.
The 40 chemical standards (1 mg) were accurately weighed and dissolved in methanol to obtain the stock standard solutions. The mixed working solution was prepared by adding 10 µL of the stock standard solution into a 1 mL volumetric flask.
Mass spectrometric analysis was operated in negative ion and positive ion modes using an ESI, and the full scan mass spectral data were collected over a range from m/z 100 to 1400 at a resolution of 35,000 and targeted MS 2 at a resolution of 17,500, triggered by parallel reaction monitoring (PRM). The optimum source parameters were as follows: spray voltage (−3.2 kV); spray voltage (+3.5 kV), the sheath gas flow rate (35 arb); aux gas flow rate (10 arb); capillary temperature (320 • C); heater temperature (350 • C); S-lens RF level (60); and the stepped, normalized collision energies (20%, 40%, and 60%).

Data Analysis
The UHPLC-MS data in Tojapride was processed using Xcalibur software version 4.2; the minimum peak intensity was set at 10,000. The chemical formulae for all parent and fragment ions of the selected peaks were calculated from the accurate mass using a formula predictor by setting the parameters as follows: The compounds in the same category possess identical carbon skeletons that will generate similar characteristic fragment ions. In this study, the fragment ion patterns of flavonols, flavanones, and glycyrrhizic acid were investigated using LC-MS/MS. The fragmentation pathway of flavonols and flavanones is shown in Figure 2A

Conclusion
A useful approach using UHPLC-Q-Exactive Orbitrap MS combined with the PRM technique was established in the present study as an effective tool for the assessment of the chemical composition of Tojapride. As a result, a total of 222 compounds were identified and 218 were isolated for the first time from Tojapride. The methods are simple, rapid, and sensitive, and provide useful MS/MS 2 data. The research results not only extend our understanding of the chemical constituents of Tojapride in the existing study, but also exhibit a wide application for the characterization and profiling of compounds in different samples.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27196639/s1, Table S1: Retention times and mass spectral data of Tojapride; Table S2: Detailed information of the 40 standards in Tojapride.
Author Contributions: L.Z. and S.Q.-investigation writing-original draft preparation; S.T., S.E., K.L., J.L. and W.C.-data processing; L.S. and H.L.-review and editing, conceptualization, and supervision. All authors have read and agreed to the published version of the manuscript.
Funding: This work was financially supported by the National Natural Science Foundation of China (no. 82004355) and the Fundamental Research Funds for the Central public welfare research institutes (ZZ14-YQ-005).