Rapid Identification of Constituents in Cephalanthus tetrandrus (Roxb.) Ridsd. et Badh. F. Using UHPLC-Q-Exactive Orbitrap Mass Spectrometry

Cephalanthus tetrandrus (Roxb.) Ridsd. et Badh. F. (CT) belongs to the Rubiaceae family. Its dried leaves are widely used in traditional Chinese medicine to treat enteritis, dysentery, toothache, furuncles, swelling, traumatic injury, fracture, bleeding, and scalding. In order to further clarify the unknown chemical composition of CT, a rapid strategy based on UHPLC-Q-exactive orbitrap was established for this analysis using a Thermo Scientific Hypersil GOLDTM aQ (100 mm × 2.1 mm, 1.9 µm) chromatographic column. The mobile phase was 0.1% formic acid water–acetonitrile, with a flow rate of 0.3 mL/min and injection volume of 2 µL; for mass spectrometry, an ESI ion source in positive and negative ion monitoring modes was adopted. A total of 135 chemicals comprising 67 chlorogenic acid derivatives, 48 flavonoids, and 20 anthocyanin derivatives were identified by comparing the mass spectrum information with standard substances, public databases, and the literature, which were all discovered for the first time in this plant. This result broadly expands the chemical composition of CT, which will contribute to understanding of its effectiveness and enable quality control.


Introduction
Cephalanthus tetrandrus (Roxb.) Ridsd. et Badh. F. (CT), known as Ma Yanshu or Water Yangmei in traditional Chinese medicine (TCM) and 'Bagua Maple' in Dong medicine, belongs to the Rubiaceae family. The leaves have the effects of clearing away heat and toxic materials, as well as dispelling blood stasis and reducing swelling, and they are used for the treatment of enteritis, dysentery, toothache caused by acute gingivitis and acute pulpitis, furuncles, swelling, traumatic injury, fracture, bleeding, and scalding [1]. This plant is mainly distributed in Hunan, Guangdong, Hainan, and Taiwan provinces. However, no detailed studies on the material basis of its medicinal effects have been reported. To date, most studies on CT have focused on its ornamental value. A previous study reported on its pharmacognosy [2]. Therefore, it is necessary to clarify the unknown chemical composition of CT.
In order to further analyze and discover its chemical composition and pharmacological effects, it is necessary to find a suitable analytical technique to analyze the complex chemical composition in CT. There are many methods for analyzing herbal medicine, including      Likewise, compounds 13 and 32 were confirmed as procyanidin B1 and procyanidin B2.    [12,13], they were identified as trans-3-O-p-coumaroylquinic acid (pCoQA), trans-4-pCoQA, cis-4-pCoQA, and cis-5-pCoQA, respectively, according to their retention time and base peak ion in the MS 2 spectrum. were identified as trans-3-SQA, cis-3-SQA, trans-5-SQA, and cis-5-SQA through comparison with the literature data [14].

Identification of Speculative Anthocyanins
The  [26]. was stored at the School of Pharmaceutical Sciences, Hunan University of Medicine, Changsha, China.

Reference Standards and Sample Preparation
The dried powder of CT (5 g) was extracted under reflux in 100 mL of 70% aqueous ethanol for 1 h, and then the extracted solution was filtrated and dried under reduced pressure to yield a brown residue, which was dissolved in methanol. The sample was centrifuged at 12,000 rpm for 20 min. A volume of 2 µL was injected into an UHPLC-Qexactive orbitrap MS for analysis. All reference standards were accurately weighed and dissolved in methanol before storing in a refrigerator at 4 • C until further analysis.

Instruments and Conditions
The instruments used for this study included a Thermo Q-exactive focus orbitrap MS connected to a Thermo Scientific Dionex Ultimate 3000 RS (Thermo Fisher Scientific, Carlsbad, CA, USA). Separation was performed on a Thermo Scientific Hypersil GOLD TM aQ (100 mm × 2.1 mm, 1.9 µm). The column temperature was kept at 35 • C, and the sample was maintained at 10 • C. The mobile phase was water with 0.1% formic acid (A) and acetonitrile (B). The gradient program was as follows: 0 min, 5% B; 2 min, 10% B; 5 min, 20% B; 10 min, 25% B; 12 min, 55% B; 20 min, 80% B; 25 min, 95% B; 26 min, 5% B; and 30 min, 5% B. MS analysis was performed in both positive and negative ionization modes using electrospray ionization (ESI) in the scan range of m/z 120-1000 at a resolution of 35,000. The source conditions were as follows: sheath gas, 30; auxiliary gas, 10; spray voltage, 3.0 kV for (−)-ESI and 3.5 kV for (+)-ESI; capillary temperature, 320 • C; auxiliary gas heater temperature, 350 • C. The MS 1 spectra were acquired in full MS mode at a resolution of 35,000, whereas MS 2 spectra were obtained by ddMS 2 or parallel reaction monitoring (PRM) mode triggered by inclusion ions [12]. The NEC (normalized collision energy) was set as 30%, with 5.0 × e 5 of the automatic gain control (AGC) target. Data were processed using Xcalibur™ version 4.1 and Compound Discoverer 3.0 (Thermo Fisher Scientific, Carlsbad, CA, USA).

Prediction of Expected Compounds
It is widely known that chemical constituents in the same category possess an identical carbon skeleton and homologous biosynthetic pathways. CGA analogues constitute a large family of esters formed between quinic acid or shikimic acid and one to four special residues, most commonly p-coumaric acid, caffeic acid, sinapic acid, and ferulic acid. Therefore, the molecular structure of the CGA derivatives can be predicted [11,26]. Likewise, flavonoids and anthocyanins can also be predicted. Quercetin and kaempferol are the carbon skeletons of flavonoids connected by hydroxyl (OH) and glycoside bonds; their structures differ in terms of the type and number of sugar units, e.g., glucose (C 6

Conclusions
In this study, a rapid and effective method for identifying the chemical constituents of CT was developed using UHPLC-Q-exactive orbitrap combined with PRM; the compounds were predicted using DFI and NL techniques. A total of 135 compounds were identified, comprising 67 chlorogenic acid derivatives, 48 flavonoids, and 20 anthocyanins, all of which are reported for the first time in CT. These results expand the knowledge on the chemical composition of CT and provide a scientific basis for the subsequent elucidation of the medicinal substances present and their activities, enabling further development and utilization of this plant. Overall, the results lay the foundation for in-depth research on the pharmacodynamic basis of CT. Furthermore, this research strategy can be used for the characterization of various samples.

Conclusions
In this study, a rapid and effective method for identifying the chemical constituents of CT was developed using UHPLC-Q-exactive orbitrap combined with PRM; the compounds were predicted using DFI and NL techniques. A total of 135 compounds were identified, comprising 67 chlorogenic acid derivatives, 48 flavonoids, and 20 anthocyanins, all of which are reported for the first time in CT. These results expand the knowledge on the chemical composition of CT and provide a scientific basis for the subsequent elucidation of the medicinal substances present and their activities, enabling further development and utilization of this plant. Overall, the results lay the foundation for in-depth research on the pharmacodynamic basis of CT. Furthermore, this research strategy can be used for the characterization of various samples.  Data Availability Statement: Data will be provided upon request.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.