Introduction

The efficacy of traditional Chinese medicines (TCMs) has been extensively validated [1]. However, obtaining the overall chemical information of numerous TCM components remains a major challenge. Therefore, establishing a strategy for rapid and comprehensive identification of chemical ingredients in TCMs is critical.

Tribulus terrestris is an annual creeper herb that grows worldwide, especially in the North temperate zones. The fruit of T. terrestris is a TCM termed “Ji Li,” which possesses various activities, including activities against heart diseases [2], eye inflammation, skin irritation [3], abdominal distention, etc. T. terrestris has been extensively used in clinical practice in China many years. However, there are some significant differences in its therapeutic effects between domestic and foreign countries. In Europe and the USA, it is mainly used for increasing muscle strength [4] and improving sexual function [5], whereas it is always used to treat cardiovascular disease [6] in China. The differences in therapeutic functions of T. terrestris could be related to their different habitats. T. terrestris contains phenolic compounds [7], saponins [8], sterols [9], flavonoids [10, 11], alkaloids [12] etc., and the chemotypes are different in T. terrestris from Southeastern Europe, East Asia, and South Asia [13]. Based on previous phytochemical studies on T. terrestris from Beijing from our own laboratory [14], we choose three different areas, including Beijing, Rome, and Xinjiang to identify the chemical constituents T. terrestris in this study.

Owing to the advantages of high resolution and sensitivity, UHPLC/Q-TOF MS is used for the analyses of complex samples, including individual herbs and their components [15, 16]. However, conventional identification requires analysts to rapidly examine each individual peak, which is time-consuming, and a rapid workflow of chemical identification is urgently required.

In this study, we proposed a strategy to utilize the UHPLC/Q-TOF MS with the informatics platform of MS data for identification of multiple components of T. terrestris. The compounds were rapidly separated by ultra-high-performance liquid chromatography (UHPLC) and accurately measured by TOF mass spectrometry. Then the data was processed and analyzed by the informatics platform, which has a TCM component in-house database and the ability to automatically identify compounds [17].

A total of 82 steroidal saponins and nine flavonoids were identified or tentatively characterized from T. terrestris in Beijing. Of these, the chemical structures of 15 new components were deduced based on their characteristic MS fragmentation patterns and common neutral loss settings. In addition, the chemical ingredients of T. terrestris, from Xinjiang and Rome, were also similarly investigated. Hitherto, a total of 141 chemical components were identified from these three samples, including 35 groups of isomers and 39 of these components were identified or tentatively identified as new compounds (Table 1, Figure 1). This study established an efficient approach for the rapid identification of chemical constituents in TCMs by UHPLC/Q-TOF MSE with the informatics platform, which has the advantages of automation, accuracy, and time-saving.

Table 1 Compounds Identified from Three Samples of T. terrestris by UPLC/Q-TOF MS
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Figure 1
figure 1

The base peak ion (BPI) chromatograms of the extracts from the negative mode in T. terrestris analyzed by UHPLC/Q-TOF MSE. (a) T. terrestris from Beijing; (b) T. terrestris from Xinjiang; (c) T. terrestris from Rome. Pink numbers, triterpenoid saponins are the major components that differed between the three samples.

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Experimental

Chemicals and Herb Materials

Acetonitrile (HPLC grade) was purchased from Fisher Scientific Co. (Loughborough, UK). Distilled water was purchased from Watsons (Guangzhou, China). Formic acid (HPLC grade) was purchased from Acros Co. Ltd. (St. Louis, MO, USA). Other reagents were obtained commercially in analytical purity (Beijing, China).

The fruits of T. terrestris were collected from Beijing and Xinjiang in China and Rome, Italy in October 2015. The plant was identified by Professor Bao-lin Guo of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

Twenty-three steroidal saponin standards, Ginsenoside Rb1, and Ginsenoside Re (Figure 2) were previously isolated and their structures were confirmed by MS as well as 1D and 2D NMR spectra.

Figure 2
figure 2

The structures of 25 standard compounds

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Preparation of Herbal Extract

The dried powder of T. terrestris fruits (7 g) was extracted two times with hot 60% aqueous EtOH for 1.5 h per extraction. The combined extracts were filtered and concentrated at reduced pressure. The extract was subjected to a SP825 column, eluted with 10% aqueous acetonitrile and 95% aqueous acetonitrile to yield fractions A and B. Ten mg of fraction B was dissolved in 1.5 mL of 60% ethanol and filtered through a 0.22 μm membrane prior to use, and 2 μL was injected for UHPLC/Q-TOF MSE analysis.

UHPLC/Q-TOF MSE Analysis

UHPLC/Q-TOF MSE analysis was performed on an Acquity UPLC system (Waters Corp., Milford, MA, USA) coupled to a Synapt G2-Si MS system (Waters Corp.). A Waters Acquity UPLC HSS T3 column (100 × 2.1 mm, 1.8 μm) was used with the column temperature at 45 °C. Mobile phases were water with 0.1% formic acid (A), and acetonitrile (B). The gradient used was as follows: (0–0.5) min, 5%→15% B; (0.5–8.0) min, 15%→20% B; (8.0–14.0) min, 20%→25% B; (14.0–15.0) min, 25%→28% B; (15.0–18.0) min, 28%→35% B; (18.0–23.0) min, 35%→45% B; (23.0–27.0) min, 45%→50% B; (27.0–32.0) min, 50%→70% B; (32.0–34.0) min, 70%→80% B; (34.0–34.1) min, 80%→96% B; (34.1–36.1) min, 96% B; (36.1–36.2) min, 96%→5% B; (36.2–37.2) min, 5% B. The flow rate was 600 μL min–1. The injection volume of sample was 2 μL. The data acquisition mode was MSE. Each extract was injected twice: once for ESI+ analysis, and once for ESI− analysis; data were acquired from 50 to 1500 Da. The source temperature was 100 °C, and the desolvation temperature was 450 °C, with desolvation gas flow of 850 L h–1. Leucine-enkephalin was used as lock mass. The capillary voltage was 3 kV. At low CE scan, the cone voltage was 30 V, and the collision energy was 6 eV (trap), and 4 eV (transfer). At high CE scan, the cone voltage was 30 V, and the collision energy was 45–60 eV (trap), and 12 eV (transfer). The instrument was controlled by Masslynx 4.1 software (Waters Corp., Milford, MA, USA).

Data Analysis by Informatics Platform

Data was analyzed using software UNIFI 1.8.1 (Waters Corp., Milford, MA, USA). The maximum allowed number of peaks detected was 1000 for 2D peak detection. The peaks intensity threshold was 80 counts of high energy and 200 counts of low energy in 3D peak detection. Mass error and fragment error were both set at 10 mDa for identification, which would be exactly predicted fragments from the structure. We selected +H-H2O, +H, +Na, –e as positive adducts and +HCOO−, –H as negative adducts. Using leucine-enkephalin as the reference compound to confirm the mass accuracy, [M-H]− 554.2620 was used in negative ion and [M+H]+ 556.2766 was used in the mode of positive ion.

Results and Discussion

Identification of the Fragmentation Patterns and Retention Times of Reference Standards

Samples were tested in both positive and negative ion modes with the same LC mobile phase in order to obtain more information on the fragmentation and chromatography patterns of steroidal saponins. The reference standards were classified into six types because of different aglycones, containing: Type I (tigogenin), Type II (gitogenin), Type III (hecogenin), Type IV (diosgenin), and their furostane-type and spirostane-type saponins, respectively; Type V (Terrestrinin D), and Type VI (Terrestrinin K). To our knowledge, most steroidal saponin glycosides obtained from Zygophyllaceae were 3-O-glycosides, in which the inner sugar was usually galactose and the outer sugars were rhamnose, xylose, and glucose, respectively. The precursor ions and characteristic fragment ions of these reference standards are discussed in detail below.

In ESI−, Parvispinoside B (a furostane-type saponin of Type I) produced a deprotonated molecular ion at m/z 1213.5822 [M-H]− and an adduct ion at m/z 1259.5913 [M+HCOO]− at low CE (Figure 3a); at high CE, Parvispinoside B produced fragment ions at m/z 1213.5822 [M-H]−, 1081.5458 [M-H-Xyl]−, 919.4926 [M-H-Xyl-Glc]−, 757.4374 [M-H-Xyl-Glc-Gal]−, and 595.3840 [M-H-Xyl-Glc-Gal-Glc]− (Figure 3b).

Figure 3
figure 3

MS and MSE spectra of Parvispinoside B (peak 67). (a) (–) low CE ESI-MS spectrum; (b) (–) high CE ESI-MS spectrum; (c) (+) low CE ESI-MS spectrum; (d) (+) high CE ESI-MS spectrum

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In ESI+, Parvispinoside B produced major fragment ions at m/z 1237.5798 [M+Na]+, 1197.5909 [M+H-H2O]+, 1035.5356 [M+H-H2O-Glc]+, 903.4908 [M+H-H2O-Glc-Xyl]+, 741.4404 [M+H-H2O-Glc-Xyl-Gal]+, 579.3866 [M+H-H2O-Glc-Xyl-Gal-Glc]+, and 417.3349 [M+H-H2O-Glc-Xyl-Gal-Glc-Gal]+ at low CE (Figure 3c), and at high CE, the characteristic fragment ions at m/z 273.2204 and 255.2127 were observed because of consecutive losses of 144 (formula C8H16O2) and one molecule of water (18 Da) from the fragment ion at m/z 417.3349 (Figure 3d). The fragmentation pathways of Parvispinoside B are shown in Figure 4.

Figure 4
figure 4

Proposed fragmentation pathways and characteristic ions of Parvispinoside B and 25S-terrestrosin I

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In ESI−, Terrestrinin R (a spirostane-type saponin of Type I) produced a [M-H]− ion at m/z 1327.6170 and a [M+HCOO]− ion at m/z 1373.6226 at low CE, and then the major fragment ions were observed at m/z 1327.6170 [M-H]−, 1195.5740 [M-H-Xyl]−, 1063.5338 [M-H-2Xyl]−, 901.4794 [M-H-2Xyl-Glc]−, 755.4213 [M-H-2Xyl-Glc-Rha]−, and 593.3729 [M-H-2Xyl-2Glc-Rha]− at high CE. In ESI+, Terrestrinin R produced major ions at m/z 1351.6177 [M+Na]+, 1149.5667 [M+H-H2O-Glc]+, 723.4333 [M+H-H2O-2Glc-2Xyl]+, 577.3747 [M+H-H2O-2Glc-2Xyl-Rha]+, 415.3233 [M+H-H2O-2Glc-2Xyl-Rha-Gal]+, 273.2238 [415.3233-C8H14O2]+, and 255.2127 [273.2238-H2O]+. When the hydroxyl group substituted on the F-ring was lost, one more double bond was added to the F ring of the aglycone. This made the neutral loss 142 Da.

Parvispinoside A (a furostane-type saponin of Type II) produced the major ions at m/z 1229.5824 [M-H]−, 1275.5659 [M+HCOO]−, 1067.5242 [M-H-Glc]−, 935.4835 [M-H-Glc-Xyl]−, 773.4296 [M-H-Glc-Xyl-Gal]−, and 611.3782 [M-H-Glc-Xyl-Gal-Glc]− in ESI−. In high CE ESI+, Parvispinoside A produced fragment ions at 1253.5767 [M+Na]+, 1213.5846 [M+H-H2O]+, 1051.5322 [M+H-H2O-Glc]+, 919.4909 [M+H-H2O-Glc-Xyl]+, 757.4380 [M+H-H2O-Glc-Xyl-Gal]+, 595.3834 [M+H-H2O-2Glc-Xyl-Gal]+, 433.3304 [M+H-H2O-2Glc-Xyl-2Gal]+, 415.3192 [433.3304-H2O]+, 289.2167 [433.3304-C8H16O2]+, 271.2057 [415.3192-C8H16O2]+, and 253.1940 [271.2057-H2O]+. Comparison of Type I and Type II produced different characteristic fragmentations at m/z 433, 415, 289, 271, and 253, attributable to one hydroxyl group at C-2 position of glycone.

25S-Terrestrosin I is a furostan-type saponin of Type III, which produced a [M-H]− ion at m/z 1095.5223 at low CE in ESI−. In high CE ESI+, the fragment ions observed at m/z 917.4700, 755.4185, 593.3688, 431.3145, 317.1978, and 299.1967 were observed, attributed to loss of two glucose and two galactose residues, one formula of C6H10O2 (114 Da), and one molecule of water from [M+H-H2O]+ (m/z 1079.5273) (Figure 4).

Terrestrinin S (pseudosapogenins of Type IV) produced ions at m/z 1239.5634 [M+HCOO]−, 1193.5570 [M-H]−, 1061.5182 [M-H-Xyl]−, 899.4608 [M-H-Xyl-Glc]−, 737.4138 [M-H-Xyl-2Glc]−, 575.3582 [M-H-Xyl-3Glc]− in ESI−. Meanwhile, in high CE ESI+, Terrestrinin S produced fragment ions at m/z 1217.5568 [M+Na]+, 1195.5745 [M+H]+, 1063.5385 [1195.5745-Xyl]+, 901.4753 [1063.5385-Glc]+, 739.4262 [901.4753-Glc]+, 577.3747 [739.4262-Glc]+, 415.3233 [577.3747-Gal]+, 271.2091 [415.3233-C8H16O2]+, and 253.1973 [271.2091-H2O]+. The ions at m/z 415, 271, and 253 in high CE ESI+ can be considered as diagnostic ions for this type of steroidal saponin.

Type V is a type of steroidal saponins with a ketone group at C-3 position, such as Terrestrinin D. In the negative ion mode, Terrestrinin D produced a deprotonated molecular ion at m/z 605.3329 [M-H]−, an adduct ion at m/z 651.3398 [M+HCOO]−, and fragment ions at m/z 443.2798 [M-H-Glc]−. In the positive ion mode, Terrestrinin D produced major fragment ions at m/z 629.3333, 589.3398, 427.2849, and 313.2132. The characteristic fragment ions at m/z 427.2849 and 313.2132 resulted from loss of one molecule of glucosyl and one formula of C6H10O2 (114 Da) from the fragment ion at m/z 589.3398 [M+H-H2O]+, respectively.

In ESI−, Terrestrinin K (Type VI) produced major fragment ions at m/z 1389.6179 [M+HCOO]−, 1211.5686, 1049.5174, 917.4786, 755.4269, and 609.3634 which were attributed to the sequential loss of one xylose, one glucose, one xylose, one glucose, one rhamnose, and one galactose residues from the [M-H]− ion (m/z 1343.6116), respectively. In high CE ESI+, Terrestrinin K produced fragment ions at m/z 1367.6095 [M+Na]+, 449.3231 [M+H-2Xy-2Glc-Rha-Gal]+, 335.2545 [449.3231-C6H10O2]+, and 299.2367 [335.2545-2H2O]+.

Therefore, it can be concluded that the precursor ion can be identified from the low CE ESI−, and the exact mass of the saponin could determine its molecular formula. The type of aglycone in the ESI+ can also be deduced. For example, the saponin must be a furostanol saponin with the [M+Na]+ and [M+H-H2O]+ ions, and without the [M+H]+ ion. If diagnostic ions were detected at m/z 417, 273, and 255, the saponin should be sarsasaponin with no substituent. On the base of the sarsasaponin, hydroxyl groups and double bonds with the different characteristic ions should be identified. The characteristic neutral loss of 144 (C8H16O2) and 114 (C6H10O2) Da could identify whether a carbonyl is located at C-12, Moreover, 142 (C8H14O2) and 112 (C6H8O2) Da can determine whether a double bond is present in the F ring. Furthermore, at high CE ESI− and the ESI+, the analysis of its characteristic fragment ions can reveal the number, type, and carbohydrate sequence of sugar moieties. Based on the retention times of reference compounds, it can be deduced that the number and position of hydroxyl group substituent at the aglycone will affect the retention times of saponins, and also affect by the number of carbohydrate units. On a C18 column, the retention times of steroidal saponins with the 25S configuration were shorter than those with the 25R configuration [19, 20]; the retention times of steroidal saponins in which the terminal sugar was galactose were shorter than those with glucose. The mass fragmentation patterns of reference standards were summarized and proposed, respectively. With these typical fragment ions, we utilized informatics platform for automatic identification.

The Workflow for Rapid Characterization from TCMs by Informatics Platform

First, it was to supplement an in-house library on the TCM component database, including the information on component names and structures from T. terrestris based published reports. Then, after importing the acquisition data into the informatics platform, it could automatically detect and filter the data. The third step was to summarize and propose individual fragmentation patterns from reference compounds and set up characteristic fragment ions and neutral loss to screen out the components with similar MS fragment behaviors. For instance, the characteristic fragments at m/z 449, 431, 417, and 415 were set in the Common Fragment Settings. Meanwhile, the characteristic neutral loss at m/z 144, 142, 114, 112, 162, 146, and 132 were set in the Common Neutral Loss Settings. The characteristic fragments at m/z 431 and 417 are the main types of nuclear fragments in steroidal saponins of T. terrestris. The structures of E and F rings are always marked by the characteristic neutral loss at m/z 144, 142, 114, and 112. The sugar moieties are deduced by the characteristic neutral loss at m/z 162, 146, and 132. Finally, the chemical structures of the potential novel components from T. terrestris were identified or characterized (Figure 5). The advantage of this strategy mainly involves utilizing neutral losses and characteristic fragments to rapidly identify the structure of the compounds.

Figure 5
figure 5

Workflow for rapid characterization of TCMs by the informatics platform

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Characterization of Compounds in T. terrestris from Beijing by the Informatics Platform

After processing the data in the informatics platform, all components were further verified based on the fragment ions, retention times, and structures described in published reports. As a result, 91 compounds were tentatively identified or identified from the extract of T. terrestris from Beijing, including 82 saponins and nine flavonoids.

Saponins

Saponins were the major compositions identified in T. terrestris. This study mainly focused on saponins to propose a strategy to rapidly characterize components (Figure 6). In the results interface, a total of 76 saponins were automatically matched. Among them, peaks 33, 35, 61, 62, and 90a were isomers with the same deprotonated molecular ion at m/z 1079.5265 [M-H]− at low CE in ESI-. As an exception, in ESI+, only peak 90a had a protonated ion at m/z 1081.5421 [M+H]+ rather than [M+H-H2O]+ ion at m/z 1063.5385. Both peaks 33 and 35 showed the same diagnostic ions at m/z 431, 317, and 299 in the ESI+. Based on their similar fragmentation behaviors and the chromatography behaviors on a C18 column [23, 39, 61], 35 was tentatively identified as the 25R stereoisomer of 33 or its isomer. The diagnostic ions at m/z 415, 271, and 253 in the ESI+ of peaks 61 and 62 indicated that their aglycone was proto-diosgenin. With the diagnostic ions at m/z 433, 415, and 271 of 90a in the ESI+, the aglycone can be deduced as gitogenin. Therefore, based on their retention times, fragment ions and comparison with known compounds from previous reports, peaks 33, 35, 61, 62, and 90a were tentatively identified as 26-O-β-D-glucopyranosyl-(25S)-5α-furostan-12-one-3β,22α,26-triol-3-O-β-D-glucopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-galactopyranoside, 26-O-β-D-glucopyranosyl-(25R)-5α-furostan-12-one-3β,22α,26-triol-3-O-β-D-glucopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-galactopyranoside, 25S-Terrestrosin J, Terrestrosin J, and 26-O-β-D-glucopyranosyl-(25S)-5α-furostan-20(22)-en-2α,3β,26-triol-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, respectively.

Figure 6
figure 6

Strategy for rapid identification of steroidal saponins from T. terrestris from Beijing by the informatics platform

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At low CE ESI−, peak 90 showed two pairs of ions at m/z 1079.5265 [M1-H]− and 1125.5328 [M1+HCOO]−, and 1327.6199 [M2-H]− and 1373.6176 [M2+HCOO]−, which indicated that there were two compounds in peak 90. At high CE ESI+, the major fragment ions were observed at m/z 1103.5272, 941.4779, 757.4393, 595.3884, 433.3389, 415.3250, and 271.2023; and at 1311.6239, 1149.5671, 1017.5239, 885.4874, 739.4200, 577.3796, 415.3212, 271.2057, and 253.1955. Based on their retention times, fragment ions, and comparison with known compounds in the literature, they were tentatively identified as 26-O-β-D-glucopyranosyl-(25S)-5α-furostan-20(22)-en-2α,3β,26-triol-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside and Terrestrinin M, respectively.

A class of new compounds with characteristic fragmentation at m/z 431 without 317 and 299 were found in the characteristic fragments by the informatics platform (Figure 7). It could be deduced that the aglycones of these compounds were not hecogenin. The characteristic fragments of peak 41 were at m/z 431.3130, 413.3029, 287.2062, and 269.1917. In contrast to the characteristic fragments of gitogenin, the aglycone of peak 41 had one more degree of unsaturation. So, peak 41 was tentatively identified as 26-O-β-D-glucopyranosyl-(25R)-furostan-5(6)-en-2α,3β,22α,26-tetrol-3-O-β-D-galactopyranoside.

Figure 7
figure 7

The identified chemical structures of compounds in T. terrestris by UPLC/Q-TOF MS

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Flavonoids

A total of nine flavonoids were automatically matched from T. terrestris from Beijing. Based on the literatures, the major aglycones were Quercetin, Isorhamnetin, and Kaempferol in T. terrestris. According to the automatic matching function of the informatics platform, in the ESI+ at high CE, Quercetin revealed a characteristic fragment at m/z 303; with specific ions at m/z 317, it could be deduced that the aglycone was Isorhamnetin; and the characteristic fragment in 287 was caused by Kaempferol. Based on the match of sugar residues, the number and types of glycosyl groups could be deduced.

Characterization of Compounds in T. terrestris from Rome by the Informatics Platform

According to the existing method, 98 compounds were screened out in both positive and negative mode of ESI, including 84 saponins, 14 flavonoids, 35 groups of isomers, and 20 new components. There was a type of new components, which could not be matched with the characteristic fragments for aglycones. According to its characteristic fragments and related literature, these compounds were tentatively identified as a type of triterpenoid saponins, which might be explained by different therapeutic functions from different habitats. Moreover, we selected two reference samples to verify its fragmentation patterns.

In ESI−, Ginsenoside Rb1, a type of protopanaxadiol saponin, produced the major ions at m/z 1153.6084 [M+HCOO]−, 945.5457, 783.4906, 621.4366, and 459.3817 caused by consecutive losses of four glucosyls from the fragment ion at m/z 1107.5948 [M-H]−. In ESI+, the major ions were 1131.6000 [M+Na]+, and 1109.6113 [M+H]+. The characteristic fragment ions at m/z 443.3863, 425.3778, 407.3669 were attributed to successive loss of one molecule of water.

Ginsenoside Re, a type of protopanaxatriol saponin, produced [M+HCOO]− (m/z 991.5457), [M-H]− (m/z 945.5482), [M-H-Glc]− (m/z 783.4901), [M-H-Rha]− (m/z 799.4845), [M-H-Rha-Glc]− (m/z 637.4354), and [M-H-Rha-Glc-Glc]− (m/z 475.3799) in ESI−. In high CE ESI+, in comparison with Ginsenoside Rb1, Ginsenoside Re produced different characteristic ions at m/z 441.3727, 423.3627, and 405.3528 for the sequential loss of two molecules of water. After addition of 475 and 459 into the common fragment settings in the negative mode, as well as 441 and 425 in the positive mode, 10 compounds were matched by automatic matching function of the informatics platform, including three protopanaxadiols and seven protopanaxatriols.

As a result, a total of 108 chemical components were identified from T. Terrestris from Rome, including 84 steroidal saponins, 14 flavonoids, and 10 triterpenoid saponins, 35 groups of isomers, and 30 new components.

Characterization of Compounds in T. terrestris from Xinjiang by the Informatics Platform

By comparing with T. terrestris from Beijing, the sample from Xinjiang had similar chemical composition. Based on the established detection methods, the chemical composition of T. terrestris from Xinjiang could be quickly identified or characterized. After artificially processing the data of the informatics platform with the confirmation, 94 compounds were identified or tentatively identified, including 87 saponins, seven flavonoids, 32 groups of isomers, and 15 new components.

Conclusions

In this study, an analytical approach for rapid characterization of chemical ingredients in TCMs was established using UHPLC/Q-TOF MS coupled with the informatics platform. Specifically, a valid strategy for identification of components of T. terrestris from different geographical regions was proposed based on the summarized fragmentation patterns. Based on the exact mass, fragmentation behaviors, retention times of different types of compounds, and known structures in literature, a total of 141 chemical components (Figure 7) were identified from these three samples of one species, including 35 groups of isomers, and 39 of these components were identified or tentatively identified as new compounds. The study of the total components of T. terrestris from these three areas revealed that although they belonged to the same species, their compositions were very different. The main components of T. terrestris from Beijing and Xinjiang were similar, 81 common compounds, mainly including flavonoids and steroidal saponins. In contrast, the T. terrestris from Rome contained some ginsenosides, which explained the different therapeutic effects of T. terrestris from different regions. This method could rapidly, effectively, and comprehensively screen and identify multiple components from TCMs.