UHPLC‐Q‐TOF‐MS/MS Dereplication to Identify Chemical Constituents of Hedera helix Leaves in Vietnam

Hedera helix has been reported to contain a wide range of metabolites and produce many pharmacological effects. This research demonstrates the determination and evaluation of the phytochemical profiling of H. helix grown in central Vietnam. Methanolic extract of ivy had been analyzed by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC‐Q‐TOF‐MS/MS). MS, and MS/MS experiments were manipulated using both negative and positive ionization modes to provide molecular mass information and production spectra for the structural elucidation of compounds. A total of 46 compounds including 24 triterpene saponins and other compounds were successfully identified of which four established saponin structures have been reported for the first time. This study has provided a base for building a quality control of the raw materials according to the profile of triterpene saponins and assessment of pharmaceutical ingredients of H. helix planted in Vietnam.


Introduction
Hedera helix L., the common ivy, is one of the 15 species of the genus Hedera, Araliaceae family. As an evergreen dioecious woody liana [1], ivy has an intense vitality, even in the cold winter. e common ivy is a popular ornamental plant in many countries. e plant grows naturally in Western, Central, and Southern Europe, North America, and Asia [2]. Hedera helix is applied to treat overactive thyroid (hyperthyroidism), rheumatic diseases, and respiratory tract inflammation [2]. In Vietnam, ivy is grown in areas with cool climates such as Da Lat, Moc Chau, and Sapa and is mainly used for decoration. erefore, it has been little published research on its chemical composition and pharmacological effects. erefore, the identification of chemical constituents is significant in the phytochemistry study of H. helix grown in Vietnam.
A triterpene saponin's chemical structure is composed of an aglycone and sugar chain(s). In most cases, a series of saponins can be found in the same plant, with similar skeleton but slightly varied sugar chains. Due to the strong polarity and structural similarities, isolating a single saponin compound is frequently challenging. Furthermore, even with high-resolution nuclear magnetic resonance (NMR), structural elucidation of saponin is difficult, especially when the sugar chain contains more than three sugar residues [7]. As a result, a new method for quickly identifying and characterizing existing and novel structures is required.
UHPLC-Q-TOF-MS/MS has been an increasingly powerful and important technique for elucidating chemical structures [8]. UHPLC-QTOF-MS/MS is capable of accurately measuring molecular mass by giving the elemental composition of obtained ions. e technique has been widely used in analyzing complex samples due to its high resolution and sensitivity. In some previously published research, UHPLC-QTOF-MS/MS was applied to characterize chemical constituents and metabolites in medicinal herbs, and obtained considerable results. Small metabolites profiling of the Eurycoma longifolia aqueous extracts were performed using LC-MS/MS [9]. An integrated approach using UHPLC-QTOF-MS/MS was developed for the systematic analysis of 46 physalins from the crude extracts of Physalis alkekengi calyx [10]. Sun et al. identified 31 saponins in Shizhu ginseng applying UPLC-MS/MS [11]. UPLC-Q-TOF-MS/MS-guided dereplication of Pulsatilla chinensis was conducted, which resulted in the identification of 22 triterpenoid saponins (11 pairs of isomers) with four aglycone skeletons [12].
To the best of our knowledge, the UHPLC-Q-TOF-MS/ MS study of phytochemicals in the H. helix leaves extract has not been announced. e present study aims to characterize the chemical constituents, especially triterpene saponins presented in the ivy leaves planted in Vietnam. e results of this work can assist in clarifying the metabolic profile of H. helix. Acceleration of finding the new compounds and assessment of the potential ingredients from this valuable species are concerned for pharmaceutical application.

Chemicals and Reagents.
Deionized water for HPLC and HPLC grade acetonitrile, methanol, and analytical grade formic acid (≥98%) were obtained from Scharlau (Barcelona, Spain).
Two reference standards including α-hederin and hederacoside C were obtained from Sigma-Aldrich Chemical Co. (Singapore). e purity of each compound was no less than 98%. e standards were stored at 4°C before being used for analysis.

Sample Preparation. Hedera helix was collected from Da
Lat province, Vietnam, and identified by botanist Tran Huu Dang MSc, Southern Institute of Ecology, Vietnam Academy of Science and Technology. A voucher specimen (Code: NaPro.33.1019) was deposited in the Center for Research and Technology Transfer, Vietnam Academy of Science and Technology. e leaves were gently washed, allowed to air dry, and cut into fine pieces. 100.0 mg of leaves pieces was accurately weighed into a tube with a cover, and 2.0 mL of methanol-water (8 : 2, v/v) solvent was added. e sample was ultrasonicated for 10 min and then heated to 50°C for 5 min. After being centrifuged, the extract was pipetted to a 10.0 mL volumetric flask. e residue was continued on the extraction step. After five times of extraction, the solution was exactly scaled up to 10.0 mL using the solvent solution.
e sample was filtrated through a 0.45-μm filter membrane before injecting it for UHPLC-Q-TOF-MS/MS analysis. Standard solutions of α-hederin and hederacoside C were prepared in methanol at a concentration of 1000 (ppm).
An AB SCIEX X500R QTOF mass spectrometer (AB SCIEX, USA) with a Turbo V ion source was coupled with the UHPLC system. Mass data were acquired in both negative and positive electrospray ionization (ESI) modes. e MS conditions were set as follows: the ion source temperature, 500°C; curtain gas, 30 psi; nebulizer gas (GS 1), 45 psi; heater gas (GS 2), 45 psi. For the TOF MS scan, the mass range was set at m/z 70-2000. For the TOF MS/MS scan, the mass range was set at m/z 50-1500. For the negative mode, ion spray voltage was set at − 4.5 kV, the declustering potential (DP) was − 70 V, the collision energy (CE) was performed at − 20 eV, and the collision energy spread (CES) was 10 eV. For the positive mode, the ion spray voltage was set at 5.5 kV, the DP was 80 V, the CE was 20 eV, and the CES was 10 eV.
All the obtained data were processed by SCIEX OS software version 1.2.0.4122 (AB SCIEX, USA). e total ion chromatograms (TICs) of the Hedera helix extract in both positive and negative modes are shown in Figure 1

Triterpene Saponins
3.1.1. Aglycones. Hedera helix aglycones include hederagenin and oleanolic acid. A relatively abundant series of dehydrated ions and/or a small aglycone ion can readily distinguish the parent skeleton for the aglycone. In the positive mode, the diagnostic fragment ions of these two aglycones can be easily detected. e chemical structures and fragmentation pathways of the hederagenin and oleanolic acid aglycones are illustrated in Figures 2 and 3, respectively.

Sugar Chains.
e sugar chains of triterpenoid saponins generally substitute at C-3 and/or C-28 position(s) of an aglycone. e common monosaccharide moieties of the sugar chains were glucopyranosyl (Glc), rhamnopyranosyl (Rha), arabinopyranosyl (Ara), and glucuronopyranosyl (Glu). e composition of sugar chains can be inferred in the positive ion mode using the characteristic fragment ions, specifically as follows: the loss of Glc is 162 Da, Rha is 146 Da, Ara is 132 Da, and Glu is 176 Da. e sugar moieties at C-3 and C-28 were eliminated successively from C-3 to C-28 and from end to inner [12].
In the negative ion mode, the typical solvent adducts ion [M+HCOO] − and deprotonated ion [M-H]− can be usually observed, which provides the molecular mass and chemical formula of a compound. Typically, the sugar chain at C-28   Journal of Analytical Methods in Chemistry tends to be completely eliminated; then, the positions and composition of oligosaccharides chains can be readily differentiated and followed by an abundant fragment ion as a base peak [12]. e major fragment ions observed in the mass spectra of the two triterpene saponins are summarized in Table 1. e typical MS and MS/MS spectra of α-hederin and hederacoside C are shown in Figure 4.

Structural Characterization of Triterpene Saponins.
Based on the earlier strategy, 24 triterpene saponins were tentatively identified and characterized from the H. helix extract. e chemical structures are illustrated in Figure 5, and the MS data are listed in Table 2.
At T R � 11.11, in the negative mode, compound 14 yielded an [M+HCOO] − ion at m/z 1295.6279 and provided fragment ions at m/z 779 and 469 corresponding to the loss of 2 Glc and 1 Rha at C-28, and a Rha-Glc sugar chain at C-3 of the hederagenin aglycone. Hence, compound 14 was Compound 16 (T R � 11.31 min) yielded an [M+HCOO] − ion at m/z 1149.5707 in the negative mode, primarily fragmented into ions at m/z 633 and 469, indicating hederagenin aglycone lost a Rha-Glc-Glc sugar chain at C-28, and a Glc at C-3. us, compound 16 was conditionally characterized as In the positive mode, the MS/MS spectra of compounds 17 (T R � 12.43 min) and 20 No. Aglycones Hederagenin 28-O-β-D-glucopyranoside                     In the positive mode, the MS/MS spectra of compounds 28

Conclusions
In the present work, by applying UHPLC-Q-TOF-MS/MS in both positive and negative electrospray ionization modes as an efficient analytical method, the chemical constituents of H. helix could be rapidly discovered and identified in a single sample injection. As a result, 46 phytochemicals including 24 triterpene saponins were characterized, and four of which have yet been published before. UHPLC-Q-TOF-MS/MS serves as a powerful analytical method for finding and instructing new phytochemical structures. Furthermore, the phytochemical profile result provides a base for quality control of H. helix raw materials. It also propels the medicinal application of this plant base on the metabolomic profiling of triterpene saponins.

Data Availability
e data used to support the results of this study are included within the article. Any further information is available from authors upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.