Diagnostic Fragment-Ion-Based for Rapid Identification of Chlorogenic Acids Derivatives in Inula cappa Using UHPLC-Q-Exactive Orbitrap Mass Spectrometry

Inula cappa (Buch.-Ham. ex D. Don) DC has been used in traditional Chinese medicine to treat malaria, dysentery, and hepatitis. Previous studies have shown that chlorogenic acid is the effective ingredient of plants in this family. And the research of the chlorogenic acid in Inula cappa will help to further improve the effective resource utilization rate of this plant. Therefore, it is necessary to establish an accurate method to characterize the chlorogenic acid components in Inula cappa. In this study, a simple, fast, and sensitive UHPLC-Q-Exactive Orbitrap mass spectrometry method was established, which can simultaneously analyze known and unknown ingredients in a short time (within 30 minutes) in Inula cappa. According to the diagnosis fragmentation ions, retention time, and bibliography, 68 chlorogenic acid derivatives were identified in Inula cappa. The results of this experiment lay the foundation for the active substances and quality control of Inula cappa and provide a theoretical basis for whether Inula cappa can be an alternative to the endangered wild medicinal materials of the same family.


Introduction
Traditional Chinese medicine (TCM) has been applied for thousands of years in China and its surrounding areas. For a long time, TCM has attracted worldwide attention because of its extremely effective treatment of certain diseases and minimal side effects. It was estimated that more than 1.5 billion people all over the world trust in the efficacy of TCM and rest assured of its safety [1]. However, it is still unclear which of the ingredients of TCM are effective for the treatment of diseases because they are a complex mixture of hundreds of different chemical components. It not only hinders the clinical promotion and application of TCM but also greatly hinders the development of TCM. In recent years, with the continuous improvement and advancement of analytical techniques and methodology, the research speed of TCM has been greatly accelerated.
Based on the diversity and complexity of the chemical components of TCM, it is necessary to use advanced analytical techniques and methods to explain the pharmacological basis and mechanism of action of TCM because ultrahigh performance liquid chromatography-mass spectrometry (UHPLC-MS) with high sensitivity is to reduce sample complexity [2]. In addition, UHPLC-MS also has the advantages of good specificity, short analysis cycle, and good signal reproducibility [3,4]. UHPLC-HRMS has now been widely used in the analysis and identification of TCM components, especially most small molecules. the whole plant have traditionally been used as medicines for treatment of malaria, dysentery, and hepatitis [4]. e compounds responsible for the various pharmacological effects of Inula cappa consist of sesquiterpene lactones (isoalantolactone and germacranolide), triterpenoids (lupeol, oleanolic acid, and β-sitosterol), steroids, anthraquinones, flavonoids (luteolin, apigenin, and chrysoeriol), fragrances, amides, and chlorogenic acid [5,6].
Chlorogenic acids (CGAs) are the family of esters phytochemicals formed between cinnamic acid derivatives and (−)-quinic acids. In recent years, chlorogenic acid have been proven to mediate for its hepatoprotective, choleretic, antimicrobial activities, antioxidant, hypoglycaemic, and antiviral activities; besides, basic and clinical investigations have implied that the consumption of chlorogenic acid can reduce the relative risks of type 2 diabetes, obesity, and Alzheimer's disease [7,8].
In this study, a simple, fast, and sensitive UHPLC-Q-Exactive Orbitrap mass spectrometry method was established, based on accurate mass, which can simultaneously analyze the known and unknown components in Inula cappa in a short time (within 30 minutes).
First, extracting the CGAs from the Inula cappa by refluxing, a high-resolution mass spectrum was obtained by using UHPLC-Q-Exactive Orbitrap MS in the negative mode and parallel reaction monitoring. Finally, the diagnostic fragment ions, retention time, and bibliography were established to process the ion chromatograms, and 68 compounds in the Inula cappa were rapidly identified.

Materials.
e decoction pieces of Inula cappa (dried root) were purchased from Bozhou Jianzheng Trading Co., Ltd., (Henan, China), which were grounded into powder before sample preparation. e voucher specimen was deposited at School of Pharmaceutical Sciences, Hunan University of Medicine.

Preparation of Standard
Solutions. Accurately weigh 10 mg of each reference standard, and dissolve it in 10 mL of methanol; then, take 10 μL and dilute to 1 mL. A volume of 1 μL was injected into UHPLC-Q-Exactive Orbitrap MS for analysis.

Preparation of Sample Solutions.
e dried powder of Inula cappa (10 g) was reflux-extracted in 50 mL 70% aqueous ethanol for 1 h, and then, the extracted solution was filtered and dried by rotary evaporation and then reconstituted with 10 ml methanol. A volume of 2 μL was injected into UHPLC-Q-Exactive Orbitrap MS for analysis.
All samples were analyzed in the negative mode as the following tune method. e nitrogen (purity ≥ 99.99%) served as sheath gas and auxiliary gas at the flow rate of 30 and 10 (arbitrary unit), respectively; the capillary temperature is 320°C; the auxiliary gas heater temperature is 350°C; and spray voltage is 3.5 kV. High-resolution mass spectrum was acquired at full scan in a mass range of m/z 120-1000 at a resolution of 35000 detected by the Orbitrap analyzer. e MS 2 data at a resolution of 17500 were obtained by the parallel reaction monitoring mode triggered by the inclusion ions list. e nitrogen (purity ≥ 99.999%) served as collision gas to generate the fragment ions, and the energy was set as normalized collision energy 30% [9].

2.7.
e Establishment of Diagnosis Fragmentation Ions. Chlorogenic acid (CGA) is a series of ester phytochemicals formed between caffeic acid and quinic acid. It is easy to understand that all CGAs use quinic acid as the backbone and produce similar fragments, which can be defined as diagnostic ions. By using diagnostic ion information, CGAs can be quickly screened and characterized. e fragmentations behavior of CGAs has been reported and summarized in the previous literature.

Data
Processing and Analysis. LC-MS data analysis was performed using Xcalibur software version 4 ( ermo Fisher Scientific, San Jose, California, USA). e raw data including the full-scan MS and MS 2 data were processed by the Compound Discover 3.0 using the expected compounds predicted method [11] based on the metabolism workflow templates to detect the chlorogenic acid derivatives constituents of Inula cappa. Finally, candidates for CGA were characterized based on the diagnostic fragment ions, retention time, and bibliography.

Conclusions
In this study, 68 chlorogenic acid and derivatives exhaustively characterized from the extract of Inula cappa by using the strategy for the rapid detection and identification of CGAs using UHPLC-Q-Exactive Orbitrap mass spectrometry with diagnostic fragment ion technology was proposed. e developed strategy was proved reliable and efficient in rapid discovery of new CGA class from Inula cappa. e results will provide new insights into the effective substances and quality control of Inula cappa. At the same time, the yield of Inula cappa is very high, and the study of the chlorogenic acid can be used as a basis to judge whether it can be used as a substitute for endangered wild medicinal materials. Besides, it also provides new insights for understanding the qualitative characteristics of these phytochemical components in other TCM.

Data Availability
e data used to support the finding of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.