Rapid Screening of Chemical Constituents in Rhizoma Anemarrhenae by UPLC-Q-TOF/MS Combined with Data Postprocessing Techniques

Rhizoma Anemarrhenae, a famous traditional Chinese medicine (TCM), is the dried rhizome of Anemarrhena asphodeloides Bge. (Anemarrhena Bunge of Liliaceae). The medicine presents anti-inflammatory, antipyretic, sedative, and diuretic effects. The chemical constituents of Rhizoma Anemarrhenae are complex and diverse, mainly including steroidal saponins, flavonoids, phenylpropanoids, benzophenones, and alkaloids. In this study, UPLC-Q-TOF/MS was used in combination with data postprocessing techniques, including characteristic fragments filter and neutral loss filter, to rapidly classify and identify the five types of substances in Rhizoma Anemarrhenae. On the basis of numerous literature reviews and according to the corresponding characteristic fragments produced by different types of compounds in combination with neutral loss filtering, we summarized the fragmentation patterns of the main five types of compounds and successfully screened and identified 32 chemical constituents in Rhizoma Anemarrhenae. The components included 18 steroidal saponins, 6 flavonoids, 4 phenylpropanoids, 2 alkaloids, and 2 benzophenones. The method established in this study provided necessary data for the study on the pharmacological effects of Rhizoma Anemarrhenae and also provided the basis for the chemical analysis and quality control of TCMs to promote the development of a method for chemical research on TCMs.


Introduction
Rhizoma Anemarrhenae is one of the traditional Chinese medicines (TCMs) that are commonly used for clearing heat and purging fire, nourishing yin, moistening dryness, reducing anxiety and thirst, and promoting excretion. This TCM is the dried rhizome of the Liliaceae Anemarrhena genus and has been widely used for clinical treatment of febrile disease, high fever and thirst, cough and asthma, osteopyrexia and fever, and other diseases [1,2]. The pharmacologically active components of Rhizoma Anemarrhenae include steroidal saponins, flavonoids, phenylpropanoids, alkaloids, and benzophenones, with saponins being the major constituents [3][4][5]. At present, the analytical methods of TCM ingredients mainly include thin layer chromatography (TLC), gas chromatography (GC), high-performance liquid chromatography (HPLC), and ultraperformance liquid chromatography/timeof-flight mass spectrometry (UPLC-Q-TOF/MS), among others. As a comprehensive analysis technique, UPLC-Q-TOF/MS has been increasingly applied for the qualitative and quantitative analysis of components in TCMs and compound preparations [6][7][8][9][10][11]. However, this method displays several disadvantages, including complicated and time-consuming data processing. Therefore, a newly developed method to rapidly identify components in TCMs is urgently needed to improve the efficiency and accuracy of the qualitative analysis and to promote the development of modernization and internationalization of TCMs.
With the rapid development of modern technology, data postprocessing techniques, mainly including characteristic fragments filter (CFF) and neutral loss filter (NLF), play increasingly important roles in TCM research [12][13][14][15][16][17]. CFF is based on compounds with the same or similar mother nucleus structures that can produce characteristic fragments to determine a certain type of compound under the same mass spectrometric conditions. NLF is based on a certain 2 Evidence-Based Complementary and Alternative Medicine type of compounds that frequently lose the same neutral fragments by energy collision splitting. According to the loss of these neutral fragments, substances with a class of characteristic substituted groups can be briefly classified [18][19][20][21][22]. CFF is favorable for the rapid screening of similar compounds, whereas NLF helps further confirmation of compounds. Thus, the combination of both techniques markedly reduces qualitative analysis difficulty and was gradually applied in the field of pharmaceutical analysis.
In recent years, UPLC-Q-TOF/MS is widely used to analyze constituents in Rhizoma Anemarrhenae [23,24]. In this study, Rhizoma Anemarrhenae was selected as an example to establish an efficient approach for the rapid screening of chemical components by using UPLC-Q-TOF/MS in combination with data postprocessing techniques. First, through reading and integration of abundant information from the literature, we summarized the fragmentation patterns of five kinds of compounds (steroidal saponins, flavonoids, phenylpropanoids, alkaloids, and benzophenones) in Rhizoma Anemarrhenae. Secondly, a full spectrum scan was performed by UPLC-Q-TOF/MS. Finally, the compounds were characterized based on the CFF and NLF rule. Compared with traditional methods, the study could rapidly classify and identify a certain type of compounds. The method of UPLC-Q-TOF/MS coupled with data postprocessing techniques not only partly overcame the difficulty of rapid classification and identification of complex components of TCM but also laid the foundation for the rapid development of composition identification and provided an analytical method for the further research and development of TCMs.

Sample Preparation. Prepared
Rhizoma Anemarrhenae was accurately weighed (5 g) and extracted twice with 50 and 40 mL of 75% methanol, respectively, and each reflux time was 1 h. The extracts were filtered, merged, and then concentrated to 0.01 g/mL. The concentrated solution was filtered using a 0.22 m syringe filter, with 2 L injected for UPLC-Q-TOF/MS analysis.
UPLC was coupled to a Q-TOF-MS system (Waters, USA) equipped with an electrospray ionization source (ESI) for scanning samples in negative ion modes. Ultrahigh purity helium (He) was used as the collision gas, and high-purity nitrogen (N 2 ) was used as nebulizing gas. The conditions of ESI source were as follows: drying gas temperature: 325 ∘ C; drying gas flow rate: 10 mL min −1 ; desolvation gas flow: 600 L/h; capillary voltage: 2.0 kV; collision energy: 20-40 eV; scan spectra from m/z 50-1500; nebulizing gas pressure: 350 psi. Leu-enkephalin ions at m/z 556.2771 and 554.2615 were used to ensure accuracy in spectral acquisition.

Data Analysis.
Original data were analyzed using Masslynx (Waters, USA) software 4.1. to detect and align the peaks. Data were processed and converted to an Excel format containing complete information on mass, retention time, and peak area of the samples. Target compounds were obtained by output data processing.

Method Development.
The chemical constituents of Rhizoma Anemarrhenae are complex and diverse, but similar kinds of compounds exhibit certain similarities in chemical structures. We summarized the fragmentation patterns of compounds in Rhizoma Anemarrhenae according to the mass spectrometric behavior; that is, the same type of compounds with the same or similar public skeleton usually show the same fracture mode to produce the same CFs. The method that uses CFs to determine a certain type of compounds is called CFF. For example, phenylpropanoid compounds can produce characteristic fragment ions at m/z 107 [C 7 H 7 O] − and 91 [C 7 H 7 ] − in negative ion mode. The ions could be screened from the total ion current chromatograms according to their CFs and then identified according to specific fragments and molecular ion peaks. In MS collision-induced dissociation, the differences between molecular ion peaks and high mass-to-charge ratio fragment peaks also play an important role in component identification. The method that uses NLs to screen compounds is called NLF. Mangiferin could easily lose neutral fragments at m/z 90 (C 3 H 6 O 3 ) and 120 (C 4 H 8 O 4 ) and could be filtered according to the two characteristic neutral fragments. CFF and NLF perform an increasingly important role in TCM research. Therefore, the complex components in Rhizoma Anemarrhenae can be rapidly and accurately classified and identified by combining UPLC-Q-TOF/MS with data postprocessing techniques (CFs and NLs). Finally, Based on the results of our study and those described in relevant and previous experiments, the rules of CFF and NLF regarding the five categories were established (shown in Table 1).
A full scan was conducted in both positive and negative modes to analyze the Rhizoma Anemarrhenae extract, and results show that the ESI negative ion mode was simpler, more stable, and easier to interpret compared with the positive ion mode, and the characteristic ions obtained in the negative ion mode could be evidently observed to distinguish the two types of steroidal saponins. Thus, the negative ion mode was used in this study. The full scan MS chromatograms of substances in Rhizoma Anemarrhenae extracts are shown in Figure 1. The fragmentation patterns of the five types of main Evidence-Based Complementary and Alternative Medicine 3  constituents in Rhizoma Anemarrhenae were summarized, and 32 compounds were identified (shown in Table 2).

Steroidal Saponins.
Certain cleavage law exists under a certain MS condition for steroidal saponins. Steroidal saponins can be divided into two types: furostanol glycosides, which bear an open side chain at C-22, and spirostanol glycosides, which contain a closed spiroketal ring at C-22. Based on abundant fragment ions generated by losing sugar chains, side chains and dehydration steroidal saponins were divided into types I to V according to the numbers of hydroxyl group and double bond in the A, B, C, and D rings [23,24,27,30].

Isomangiferin
Mangiferin-type flavonoids [ Furostanol glycosides [5,27] Evidence-Based Complementary and Alternative Medicine 5 Spirostanol glycosides [27] 6 Evidence-Based Complementary and Alternative Medicine    Table 1, compound 29 was revealed as spirostanol saponins (type I). These results are consistent with the fragmentation pathway of timosaponin AIII [5,27]. A proposed mechanistic pathway for fragments formed in MS is shown in Figure 3.

Flavonoids.
Flavonoids were divided into mangiferintype flavonoids, chalcone-type flavonoids, flavanones, homoisoflavonoids, and icariin-type flavonoids according to their chemical structures. Mangiferin-type flavonoids shared the same aglycone structure, the aglycone and sugar of which formed C-glycosides, and C-glycosides tended to lose CH 2 O units during MS, and 2/3 and 3/4 CH 2 O units could be eliminated from sugar molecules through 0,3 X/ 0,2 X cleavage [5,25]; therefore, under the negative ion mode, the C-glycosides easily lost the neutral fragments at m/z 90 (C 3 H 6 O 3 ) and 120  Table 1. Compound 1 was rapidly and efficiently revealed as mangiferin-type flavonoids. Therefore, compound 1 was determined to be neomangiferin [5,25]. The specific fragmentation process of neomangiferin is shown in Figure 4. chalcone flavonoids. Finally, compound 22 was identified as 2 ,4 ,4-trihydroxychalcone [29]. The specific fragmentation process of 2 ,4 ,4-trihydroxychalcone is shown in Figure 5.   Table 1, compound 28 shared the same fragmentation pathways with compound 27 and belonged to coumarin. Therefore, compound 28 was determined as hinokiresinol [29,33]. A pathway for fragments formed during MS is shown in Figure 6.

Benzophenones.
Benzophenone compounds possess the same A ring structure, which easily loses 94 Da (A ring) and 44 Da (CO 2 ) in the negative ion mode. Therefore, benzophenone compounds in Rhizoma Anemarrhenae could be rapidly screened and identified according to the characteristics neutral loss of 94 Da and 44 Da in combination with molecular ion [26,34].  Table 1, and the compound was identified as benzophenone. Besides, m/z 259 [M-H] − is the parent ion; thus, compound 12 was determined as 2,6,4 -trihydroxy-4methoxybenzophenone [26,34]. A pathway for fragments formed in MS is shown in Figure 7.
3.6. Alkaloids. Alkaloids were divided into amide alkaloids with a N-C=O structure and pyridine alkaloids with N atoms in a six-member ring. In the negative ion mode, amide alkaloid with the same part of the structure easily loses a tyramine residue, resulting in neutral loss of molecule 119 (C 8 H 7 O). In addition, 15 Da (CH 3 ) can be easily lost based on the parent ion in the high mass-to-charge ratio. Given the same pyridine ring structure of pyridine alkaloids, the characteristic fragment ion at m/z 78 [C 5 H 4 N] − was produced by removing the side chain to form the pyridine ring structure, and compounds were further identified through molecular ions [28,35]. Compound 10 presented a retention time of 6.86 min and a formula of C 18 H 19 NO 4 and exhibited several fragments   Table 1, In addition, the fragment ion at m/z 312 [M-H] − was the molecular ion. Thus, compound 10 was identified as N-trans-feruloyltyramine or N-cis-feruloyltyramine [28].