Rapid Identification of Characteristic Chemical Constituents of Panax ginseng, Panax quinquefolius, and Panax japonicus Using UPLC-Q-TOF/MS

Saponins are the main active components in Panax ginseng C. A. Mey. (PG), Panax quinquefolius L. (PQ), and Panax japonicus C. A. Mey. (PJ), which belong to the genus Panax in the Araliaceae family. Because the chemical components in the three species are similar, they are often mixed and misused in functional foods and pharmaceuticals applications. Therefore, it is urgent to establish a method to quickly distinguish among PG, PQ, and PJ. Ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was combined with data postprocessing to identify the main characteristic fragments (CFs) and the related neutral losses (NLs) of protopanaxadiol (PPD), protopanaxatriol (PPT), oleanolic acid (OLE), and ocotillol- (OCO-) type saponins. By comparing the mass spectral data, it was possible to rapidly classify and identify saponins in PG, PQ, and PJ. A total of twenty-three chemical components were identified in the PG samples, twenty-three components were identified in the PQ samples, and twenty-seven components were identified in the PJ samples. Among them, OCO-type saponins were characteristic of PQ and PJ. Ginsenoside Rf, which was absent from PQ, allowed for differentiation between PQ and PJ. The CFs and NLs in the mass spectra of the characteristic components of PG, PQ, and PJ allowed for the rapid classification and identification of these species. Additionally, these results provide technical support for the quality evaluation of Chinese herbal medicine and for constructing a scientific regulatory system.


Introduction
Based on their morphology, these plants can be divided into two groups: the first is an upright rhizome with developed fleshy roots, mainly containing dammarane-(DAM-) type tetracyclic triterpenoid saponins, such as PG, PQ, Panax notoginseng, and so on . e other is a developed rhizome, properties [2][3][4][5][6]. Consequently, ginsenosides are widely used in food, healthcare products, cosmetics, and medicine. Although the three traditional Chinese medicinal herbs from the genus Panax have different pharmacological actions, indications, and clinical applications, the properties and chemical composition of these Chinese herbal species are very similar, and thus adulterated products are often passed off as genuine in the market [7][8][9][10]. For example, in order to reduce the production cost or simply via mistaken identity, PQ is added to commercial PG products [11], and narrowleaf Panax japonicus and Panax notoginseng of the same or different families and genera are often used as adulterants intentionally or mistakenly as a substitute for genuine PJ [12]. Adulterants not only compromise the integrity of the Chinese herbal medicine market but also affect the efficacy and safety of traditional Chinese medicine. erefore, it is urgent to establish methods for the rapid identification of the three genuses of Panax used in traditional Chinese medicines so as to improve the efficacy of quality evaluation and provide scientific regulation. e ginsenosides found in PG can be divided into two groups according to their glycosidic structure: DAM-type and OLE-type.
ere are two types of DAM: protopanaxadiol-(PPD-) type saponins, for which the aglycone is 20(s)-PPD; these contain the most ginsenosides, including ginsenoside Rb 1 , Rb 2 , Rb 3 , Rc, Rd, Rg 3 , and Rh 2 , and protopanaxatriol-(PPT-) type saponins, for which the aglycone is 20(s)-PPT, including ginsenoside Re, Rf, Rg 1 , and Rh 1 . e aglycone of OLE-type ginsenosides, such as ginsenoside Ro, is oleanolic acid [13]. Compared to PG, PQ and PJ not only contain PPD-, PPT-, and OLE-type saponins but also contain ocotillol-(OCO-) type saponins, such as pseudoginsenoside F 11 and pseudoginsenoside RT 4 [14,15]. In addition, ginsenoside Rf has not been found in PQ [16]. e types of saponins, similar to the structures of their parental nucleus, are rich and complex. erefore, it is necessary to develop a rapid method for the qualitative analysis of saponins that allows for the accurate classification and identification of different traditional Chinese medicines from the genus Panax.
In this study, an accurate, rapid, and sensitive ultraperformance liquid chromatography quadrupole tandem timeof-flight mass spectrometry (UPLC-Q-TOF/MS) technique combined with data postprocessing is established ( Figure 1). First, the characteristic fragments (CFs) and neutral losses (NLs) of various saponins are summarized. Based on the quasimolecular ions and the fragment ions provided by high-resolution mass spectrometry, the chromatographic retention time, and related literature data, the saponin profiles of PG, PQ, and PJ are identified in order to realize accurate distinction between the three. is study aims to explore the medicinal basis of the three traditional Chinese medicinal herbs from the genus Panax and provide basic information for establishing a comprehensive system for evaluating the quality of medicinal materials. Simultaneously, this approach can provide technical support for constructing a scientifically based regulatory system.

Materials, Reagents, and Instruments.
Nine batches of representative medicinal materials were collected or purchased from Jilin, the main area producing PG and PQ, and from different areas producing PJ. e detailed sample information is presented in Table 1. High-performance liquid chromatography-grade acetonitrile was provided by Oceanpak (Sweden), high-performance liquid chromatography-grade formic acid was provided by ermo Fisher (USA), and distilled water was purchased from Watsons Food and Beverage Company (China). A Waters Acquity (Waters, USA) UPLC instrument and a Xevo G2 (Waters, USA) Q-TOF/MS system were used in this study.

Sample Preparation.
e Chinese medicinal herbs PG-1, PQ-1, and PJ-1 were, respectively, crushed, and 0.2 g of the powdered PG-1, PQ-1, and PJ-1 was placed into three separate test tubes, soaked in 10 mL of 70% ethanol, and ultrasonically extracted for 50 min. After extraction, each tube was cooled and centrifuged for 10 min. e supernatant was subsequently filtered through a 0.22 μm microporous membrane and analyzed by UPLC-Q-TOF/MS.

UPLC and MS
Conditions. UPLC conditions were as follows: a Waters Acquity UPLC BEH C18 column (2.1 mm× 100 mm, 1.7 µm) was used as the chromatographic column. e column temperature was set at 40°C, the flow rate was 0.3 mL/min, the injection volume was 5 µL, the mobile phase was composed of 0.1% formic acid aqueous solution (A) and acetonitrile (B), and the chromatographic separation was carried out by gradient elution, where the gradient sequence was as follows: 0-2 min, 5-10% B; 2-6 min, 10-30% B; 6-10 min, 30-50% B; 10-15 min,  50-80% B; 15-20 min, 80-100% B; 20-25 min, 100% B;  25-30 min, 100-5% B; and 30-35 min, 5% B. TOF-MS conditions were as follows: mass spectrometry was performed using a Waters G2 Q-TOF mass spectrometer, equipped with a negative mode electrospray ionization source. e capillary voltage was −2.4 kV, the cone voltage was 40 V, the source temperature was 120°C, the desolvation temperature was 400°C, the desolvation gas was 800 L/h, and the cone gas was 50 L/h, using leucine enkephalin (m/z 554.2615) as an external reference. In order to ensure the accuracy of the data acquisition, the full-scan data in the range of 100-1500 Da were obtained.

Method Establishment.
e main pharmacological constituents of PG, PQ, and PJ are saponins. erefore, to accurately distinguish the three traditional Chinese medicines, it was necessary to classify and identify the saponins. However, the use of conventional methods to determine the composition of saponins is complicated and time-consuming because of their large molecular weight and similar core structure. In collision-induced MS, compounds with the same or similar parent nuclear skeletons usually fracture similarly, and this technique is used to establish fragmentation patterns. CFs are molecular compounds with the same or similar parent core structures. When exposed to the energy impact of MS, they can fragment into ions, from which the cleavage type and material can be easily inferred. CFs can be used to help to rapidly classify the target materials. In addition, molecular ions can lose neutral radicals or molecules in MS, as shown by the difference between the mass/load ratio and the molecular ion peak and the product ion peaks, respectively. ese lost free-radicals or molecules are known as NLs, which aid the screening and identification of substances [17][18][19][20][21][22]. erefore, we present the MS fragmentation of PG, PQ, and PJ and summarize their CFs and common NLs, which are based on the different core structures (DAM-, OLE-, and OCO-types). First, the different CFs were used to preliminarily classify the unknown components.
e various saponins were identified by combined analysis of their molecular ions, retention time, and the fragmentation pattern of the unknown components, along with their fracture processes, which were estimated using common NLs. Based on the types of saponins in the samples, the three traditional Chinese medicinal herbs could be identified quickly and accurately.

Results and Discussion
Based on the summarized CF and NL data, PG, PQ, and PJ were analyzed. Twenty-three chemical constituents were identified for the PG samples, which included 10 PPD saponins, 11 PPT saponins, and 2 OLE saponins. A total of twenty-three components was identified from PQ, which included 12 PPD saponins, 4 PPT saponins, 3 OLE saponins, and 4 OCO saponins. A total of twenty-seven components was identified in the PJ samples, which included 7 PPD saponins, 6 PPT saponins, 11 OLE saponins, and 3 OCO saponins. e CFs and NLs of the different types of saponins are shown in Figure 2. e total ion chromatograms of the PG, PQ, and PJ extracts in negative ion mode are shown in Figure 3, and their compositions are shown in Tables 2-4.

Analysis of Dammarane-Type Saponins by MS
3.1.1. PPD-Type Saponins. PPD-type ginsenosides, such as ginsenosides Rb, Rb 2 , Rc, and Rg 3 , are saponins in the genus Panax. In 1966, Shibata et al. isolated ginsenediol from the root of ginseng for the first time and reported its chemical properties and structure [35]. Considering the structural types of PPD and the mass spectral information in the literature, it was found that two CFs were produced, with signals at m/z 621 [C 36       erefore, based on the CFs and NLs, it was possible to identify the compounds and infer their fracture processes.
Compound 11 (  Table 2  erefore, compound 11 (Table 2) was identified as malonylginsenoside Rb 1 from its molecular ion and secondary mass spectral fracture pattern [24,30]. e cleavage pathway of malonyl-ginsenoside Rb 1 in negative ion mode is shown in Figures 4    6 Journal of Analytical Methods in Chemistry  [27] Journal of Analytical Methods in Chemistry 7  Journal of Analytical Methods in Chemistry 9 10 Journal of Analytical Methods in Chemistry  [28,29] Journal of Analytical Methods in Chemistry 11  Journal of Analytical Methods in Chemistry 13

PPT-Type Saponins.
us far, PPT-type saponins, such as ginsenoside Re and ginsenoside Rg 1 , have been found in ginseng plants. Notably, PQ did not contain ginsenoside Rf. e CFs of these saponins occurred at m/z 637 [C 36 Table 4) was identified as ginsenoside Rf [31,32]. e cleavage pathway of ginsenoside Rf was as follows: the product ion peak at m/z 637.4339 was generated by the loss of one glucose residue (162 Da) from the molecular ion at m/z 799.4830. e fragment ion peak at m/z 475.3769 was generated when the product ion at m/z 637.4339 lost one glucose residue (162 Da). e fragmentation information and process for ginsenoside Rf are shown in Figures 8 and 9.

Analysis of OLE-Type Saponins by MS.
Pentacyclic triterpenoid saponins of the OLE-type are characteristic components of ginseng. ere are differences in the species and availability of different ginseng plants [36]. erefore, the OLE-type saponins could be quickly identified and described using the CF information and the retention times of the fractured C-3 and C-28 ester bases.

Analysis of OCO-Type Saponins by Mass Spectrometry.
A furan ring was introduced into the C-20 and C-24 positions of the dammarane skeleton through a connection with oxygen, which resulted in the formation of an OCOtype saponin [15].

Analysis of Differences in Saponins.
From identification of the chemical components, the characteristic absence of ginsenoside Rf in PQ was noted; on this premise, PQ can be differentiated. OCO saponins were not found in PG, which could be useful in identifying PG and PJ. Based on the information in Tables 2-4, the distribution of saponins among PG, PQ, and PJ are shown in the Venn diagram in Figure 14(a). e results show that ginsenoside Rg 1 , zingibroside R 1 , ginsenoside Re, ginsenoside Ro, and ginsenoside Rd were the common components of PG, PQ, and PJ. Based on this information, the ginsenoside content in PG, PQ, and PJ was preliminarily analyzed. e main ginsenosides (Rg 1 , Re, Rb 1 , Rc, and Rd) generally account for more than 70% of the total content of ginsenosides in PQ [37]. e common components, the characteristic components of the three traditional Chinese medicines, along with ginsenoside Rg 1 , zingibroside R 1 , ginsenoside Re, ginsenoside Rd, ginsenoside Rf, and OCO-type saponins (taking pseudoginsenoside F 11 as an example), were selected for comparison [38,39]. e contents of these six components in the nine batches of medicinal materials were analyzed (Figure 14(b)). ese results show that the common chemical components in PG, PQ, and PJ were present in significantly different contents and that characteristic components only existed in specific medicinal materials. Considering that the differences in the saponins in PG, PQ, and PJ were preliminarily analyzed,    Table 2 Rt=8. 23   future studies on the three kinds of medicinal materials from the genus Panax are needed. Nonetheless, the results have provided a foundation for the quantitative study of PG, PQ, and PJ and for screening the pharmacological components.

Conclusions
In this study, fragment ions associated with the chemical constituents in PG, PQ, and PJ were studied. Additionally, mass spectra fragmentation rules for the DAM-type (including PPD-and PPT-type), OLE-type, and OCO-type saponins were presented. e chemical constituents and different saponins from PG, PQ, and PJ were analyzed using UPLC-Q-TOF/MS. With the aid of the fragmentation rules for various components, 23 chemical components were identified in PG, 23 chemical components were identified in PQ, and 27 chemical components were identified in PJ. Among them, PG did not contain OCO-type saponins; thus, it was distinguishable from PQ and PJ. Additionally, ginsenoside Rf, a characteristic component, was not found in PQ, which provides a basis for differentiating between PQ and PJ. rough rapid classification and identification of the components, we differentiated among three types of traditional Chinese medicinal herbs from the genus Panax. is study provides a foundation for pharmacodynamic research and the development of MS in the identification of traditional Chinese medicine. us, this study presents a guaranteed approach for the determination of chemical components, along with the development and application of ginseng in traditional Chinese medicine.

Data Availability
No data were used to support this study.

Conflicts of Interest
e authors declare that there are no conflicts of interest with respect to the research, authorship, and/or publication of this article.