Article

An Innovative Strategy for Untargeted Mass Spectrometry Data

Analysis: Rapid Chemical Profiling of the Medicinal Plant
Terminalia chebula Using Ultra-High-Performance Liquid
Chromatography Coupled with Q/TOF Mass Spectrometry–
Key Ion Diagnostics–Neutral Loss Filtering
Jia Yu 1, Xinyan Zhao 2, Yuqi He 2, Yi Zhang 1,* and Ce Tang 1,*

1 School of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China;
yu77@cdutcm.edu.cn
2 School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China;

19946734998@163.com (X.Z.); 18502828016@163.com (Y.H.)

* Correspondence: zhangyi@cdutcm.edu.cn (Y.Z.); tangce@cdutcm.edu.cn (C.T.)

Abstract: Structural characterization of natural products in complex herbal extracts re-

mains a major challenge in phytochemical analysis. In this study, we present a novel post-
acquisition data-processing strategy—key ion diagnostics–neutral loss filtering (KID-
NLF)—combined with ultra-high-performance liquid chromatography–quadrupole time-
of-flight mass spectrometry (UPLC-Q/TOF-MS) for systematic profiling of the medicinal
plant Terminalia chebula. The strategy consists of four main steps. First, untargeted data
Academic Editor: Giovanni D’Orazio are acquired in negative electrospray ionization (ESI⁻) mode. Second, a genus-specific di-
Received: 7 April 2025 agnostic ion database is constructed by leveraging characteristic fragment ions (e.g., gallic
Revised: 27 May 2025 acid, chebuloyl, and HHDP groups) and conserved substructures. Third, MS/MS data are
Accepted: 30 May 2025 high-resolution filtered using key ion diagnostics and neutral loss patterns (302 Da for
Published: 3 June 2025
HHDP; 320 Da for chebuloyl). Finally, structures are elucidated via detailed spectral anal-
Citation: Yu, J.; Zhao, X.; He, Y.; ysis. The methanol extract of T. chebula was separated on a C18 column using a gradient
Zhang, Y.; Tang, C. An Innovative of acetonitrile and 0.1% aqueous formic acid within 33 min. This separation enabled de-
Strategy for Untargeted Mass
tection of 164 compounds, of which 47 were reported for the first time. Based on fragmen-
Spectrometry Data Analysis: Rapid
Chemical Profiling of the Medicinal
tation pathways and diagnostic ions (e.g., m/z 169 for gallic acid, m/z 301 for ellagic acid,
Plant Terminalia chebula Using and neutral losses of 152, 302, and 320 Da), the compounds were classified into three major
Ultra-High-Performance Liquid groups: gallic acid derivatives, ellagitannins (containing HHDP, chebuloyl, or neo-
Chromatography Coupled with chebuloyl moieties), and triterpenoid glycosides. KID-NLF overcomes key limitations of
Q/TOF Mass Spectrometry–Key Ion conventional workflows—namely, isomer discrimination and detection of low-abun-
Diagnostics–Neutral Loss Filtering.
dance compounds—by exploiting genus-specific structural signatures. This strategy
Molecules 2025, 30, 2451. https://
doi.org/10.3390/molecules30112451
demonstrates high efficiency in resolving complex polyphenolic and triterpenoid profiles
and enables rapid annotation of both known and novel metabolites. This study highlights
Copyright: © 2025 by the authors.
KID-NLF as a robust framework for phytochemical analysis in species with high chemical
Licensee MDPI, Basel, Switzerland.
This article is an open access article
complexity. It also paves the way for applications in quality control, drug discovery, and
distributed under the terms and mechanistic studies of medicinal plants.
conditions of the Creative Commons
Attribution (CC BY) license Keywords: Terminalia chebula; key ion diagnostics; neutral loss filtering;
(https://creativecommons.org/license untargeted mass spectrometry; UPLC-Q-TOF/MS
s/by/4.0/).

Molecules 2025, 30, 2451 https://doi.org/10.3390/molecules30112451

Molecules 2025, 30, 2451 2 of 32

1. Introduction
The exploration of medicinal plants has gained significant momentum globally,
driven by their long-established therapeutic history and untapped potential for drug dis-
covery. A central aspect of this effort is the thorough analysis of the chemical composition
of medicinal plants, as their efficacy, safety, and quality are inherently tied to their phyto-
chemical profiles [1]. High-resolution mass spectrometry (HRMS), particularly when com-
bined with ultra-high-performance liquid chromatography (UPLC), has become a corner-
stone technology for analyzing complex plant extracts, facilitating the rapid identification
of both known and novel compounds [2]. However, some limitations remain. For instance,
the peak capacity is limited, making it challenging to fulfill the separation requirements
of chemical components in the complex systems of medicinal plants [3]. Traditional iden-
tification strategies, which rely on chromatographic retention behavior and mass spec-
trometry cleavage rules, are constrained by a limited number of reference substances and
inadequate structural coverage in dedicated databases [4].
Current strategies for LC-MS/MS data processing are typically classified into targeted
and untargeted approaches [5]. Targeted methods rely on predefined compound libraries
and fragmentation pathways, offering high specificity but limited capacity for discovering
novel compounds. In contrast, untargeted methods capture MS/MS spectra for all detect-
able ions, providing a comprehensive chemical profile but resulting in large datasets that
require advanced post-acquisition processing. In recent years, feature-based molecular
networking (FBMN) [6] and ion identity molecular networking (IIMN) have enhanced
metabolite grouping by integrating retention time and admixture data [7]. Additionally,
tools such as SIRIUS 5, powered by deep learning, have increased annotation accuracy
through fragment tree calculations [8]. However, these general strategies remain inade-
quate for highly specific structures. These include excimer ion matching errors, automatic
matching of endogenous cleavage fragments to other components, and the low recogni-
tion accuracy of complex additive ions [9].
Terminalia chebula, known as the “king of Tibetan medicine”, has been used for thou-
sands of years in traditional Chinese and Ayurvedic medicine [10]. T. chebula is used in
the treatment of asthma, bronchitis, hepatitis, dyspepsia, eye diseases, and hoarseness and
to promote hair growth [11]. The flesh of the plant has been used to treat diarrhea, leprosy,
and edema [12]. It improves appetite, reduces cholesterol and blood pressure, strengthens
the immune system, prevents aging, and enhances resistance to infections [13]. In clinical
practice, the therapeutic effects of specific preparations can be tailored to treat various
diseases and optimized by combining them based on distinct cold and heat symptoms
[14]. T. chebula contains a diverse array of chemical constituents, primarily including phe-
nolic acids, tannins, triterpenoids, and flavonoids [15]. In T. chebula, 33% of the total phy-
toconstituents are hydrolysable tannins, with variation between 20 and 50%. These tan-
nins contain phenolic carboxylic acids such as gallic acid, as well as gallotannins. Ellag-
itannins including punacalagin, casuarinin, corilagin, and terchebulin as well as others
such as chebulanin, neochebulinic, chebulagic, and chebulinic acids are also present [16].
However, the chemical complexity of T. chebula poses a unique analytical challenge. Its
ellagic tannins often contain multiple configurations, including HHDP, chebulyl, or neo-
chebulyl groups. Numerous isomers with the same molecular weight exist, and their frag-
ment pathways are complex. Most components still lack systematic annotation, which sig-
nificantly hinders the establishment of quality control standards and the investigation of
the material basis for its efficacy. Therefore, addressing the challenge of component anal-
ysis in T. chebula can reveal the chemical nature of its therapeutic potential and offer in-
sights into the metabolomic analysis of similar complex plants.
To overcome the above limitations, combined with the additive ions that are easily
formed by the T. chebula components and the characteristic fragments, key ion
Molecules 2025, 30, 2451 3 of 32

diagnostics–neutral loss filtering (KID-NLF) is presented: a post-acquisition data pro-

cessing approach that leverages characteristic fragment ions and neutral loss patterns of
target compounds. KID-NLF constructs a genus-specific diagnostic ion database based on
known T. chebula metabolites and their fragmentation profiles. This enables the prioritiza-
tion of structurally relevant ions in complex, untargeted MS/MS data, thereby improving
both the speed and accuracy of compound identification. The approach consists of two
key components: (1) key ion diagnosis (KID), which utilizes high-resolution MS/MS data
to identify diagnostic fragment ions specific to gallic acid derivatives, ellagitannins, and
triterpenoids; and (2) neutral loss filtering (NLF), which systematically detects character-
istic mass losses (e.g., 302 Da for HHDP, 320 Da for chebuloyl) to isolate structurally re-
lated compounds. This approach addresses challenges in isomer discrimination and low-
abundance compound detection by exploiting the unique structural signatures of T.
chebula constituents. It offers a robust framework for profiling even the most complex
plant extracts.
In this study, KID-NLF was applied to analyze methanol extracts of T. chebula using
UPLC-Q/TOF-MS. The method enabled the rapid identification of 164 compounds, 47 of
which are newly reported. KID-NLF overcomes limitations of existing workflows by com-
bining targeted fragmentation analysis with genus-specific database curation. This pro-
vides a transformative strategy for phytochemical profiling, especially in species rich in
polyphenols and triterpenoids. The strategy enhances understanding of T. chebula’s chem-
ical diversity and provides a scalable framework for quality control and drug discovery
in medicinal plant research.

2. Results and Discussion

2.1. Research Strategy
Despite observing only a few major peaks in the UHPLC/UV analysis of T. chebula,
the enlarged chromatogram revealed numerous minor compounds (Figure 1). To conduct
a comprehensive analysis of these compounds, a 70% methanol extract was examined us-
ing UPLC-Q-TOF/MS in a non-targeted method (without specifying the parent ion). Op-
erating in (—)-ESI mode, the analysis detected numerous compounds, facilitating the ac-
quisition of key quasi-molecular ions with enhanced sensitivity. During the scan, all par-
ent ions underwent analysis in MSE mode at a collision energy ranging from 20 to 40 eV.
Using these settings, satisfactory secondary mass spectrometry data were obtained.
Subsequently, the data underwent processing using targeted KID-NLF efficiently
identifying the parent ion and its respective daughter ion structure. The fundamental
structure of T. chebula compounds, gallic acid, features similar skeletons and substitution
patterns that facilitate the generation of KID and NLF in MS/MS spectra. KID-NLF were
identified from the MS/MS spectra of reference substances, leading to the creation of a
database cataloguing ions related to all reported Terminalia compound structures. High-
resolution KID were next extracted via UNIFI software (Version 1.9) for MS diagnosis and
MS/MS spectral data filtering, achieving rapid and efficient structural identification. Ulti-
mately, further determination of the identified compounds’ structures was accomplished
through in-depth analysis of their high-resolution MS and MS/MS spectra.
Molecules 2025, 30, 2451 4 of 32

Figure 1. UHPLC/UV chromatograms of Terminalia chebula extract (254 nm).

In summary, the analytical strategy comprises four key steps: (1) acquisition of high-
resolution MS and MS/MS data via UPLC-Q-TOF/MS; (2) creation of a diagnostic ion da-
tabase informed by MS/MS fragments, similar skeletons, and substitution patterns of re-
ported T. chebula compounds; (3) identification of precursor ions and significant fragments
using high-resolution diagnostic ions and neutral loss filtering; and (4) further elucidation
of target compound structures through detailed MS and MS/MS spectral analysis (Figure
2). According to the KID-NLF strategy, a total of 164 components were identified (Figure
3).
Molecules 2025, 30, 2451 5 of 32

Figure 2. Construction of key ion database (A) and the identification of 164 chemical constituents in
T. chebula using the KID-NLF strategy (B). The red dashed box marks components compliant with
the KID strategy, the purple dashed box denotes those not only adhering to the KID strategy but
also aligning with the NLF rules.
Molecules 2025, 30, 2451 6 of 32

Figure 3. The representative base peak intensity (BPI) chromatogram of T. chebula in negative ion
mode. The box and arrow denote the enlarged section.

2.2. Establishing a Diagnostic Ion Database

The process for establishing the database is illustrated in Figure 2A. Initially, the lit-
erature was reviewed to summarize and classify T. chebula compounds into three main
structural types: gallic acid and its derivatives, ellagic acid and its derivatives, and terpe-
noids. Subsequently, characteristic diagnostic ions were identified for each type of com-
pound.
For gallic acid and its derivatives, diagnosis involved identifying quasi-molecular
ions, including [M−2H]2− and [M−H]− ions. These compounds frequently feature multiple
galloyl groups or exhibit neutral loss of gallic acid, resulting in ions such as [M−H−152]−
and [M−H−170]−. Consequently, the accurate masses of [M−H−152]−, [M−H−170]−, and po-
tential derivatives were utilized to develop a diagnostic ion database.
T. chebula contains ellagic acid and its derivatives, typically comprising HHDP (302
Da), chebuloyl (320 Da), neoche (338 Da), THDP (292 Da), DHHDP (318 Da), flavgallonyl
(452 Da), and Gallagyl (602 Da) groups. These groups form complex combinations,
Molecules 2025, 30, 2451 7 of 32

possibly including HHDP alongside groups such as chebuloyl, neoche, THDP, DHHDP,
Gallagyl, or flavgallonyl (Figure 4). In a similar manner, a diagnostic ion database for T.
chebula ellagic acid compounds was created using the accurate masses of potential deriv-
atives.

Figure 4. The main neutral loss fragment in T. chebula.

Terpenoids typically yield abundant deprotonated molecular ions [M−H]− in the pri-
mary mass spectrometer, with some compounds also producing [2M−H]− ions. Terpe-
noids in T. chebula commonly attach to sugar groups like glucose (Glc), galloyl, and glu-
coheptonic acid via various substitution methods. Aglycone fragments, which are rela-
tively stable, typically lose sugar and galloyl segments (152 Da, 162 Da, 208 Da) under
standard voltage. Analysis of terpenoid cleavage fragments reveals two primary types of
aglycones, 503 Da and 487 Da, each with various configurations. Ultimately, the diagnos-
tic ion database for T. chebula terpenoids was developed using the accurate masses of po-
tential sugar derivatives.

2.3. Chemical Composition Analysis of T. chebula by Key Ion Diagnostics and Neutral Loss
Filtering Using UPLC-Q-TOF/MS
Mass spectrometry analysis of gallic tannin or glucose gallate frequently reveals the
neutral loss of multiple galloyl groups and gallic esters, as evidenced by the observation
of fragments such as [M−H−152]− and [M−H−170]−. These characteristic neutral loss frag-
ments serve as valuable indicators for deducing the molecular structure. Additionally, the
primary mass spectrum typically displays high-intensity [M−2H]2− peaks for macromolec-
ular components containing multiple galloyl groups, while derivatives with fewer galloyl
groups primarily generate [M−H]− or [M+HCOO]− adduct ions. However, misinterpreta-
tion may occur wherein the [M−2H]2− peak is incorrectly identified as [M−H]−, leading to
the misidentification of the true [M−H]− peak as [2M−H]−. To mitigate the potential for
such misjudgments, the secondary mass spectrum is generally employed for confirma-
tion. A comparative analysis of the primary and secondary mass spectra reveals that frag-
ments exhibiting higher intensity in the primary mass spectrum often disappear in the
secondary mass spectrum, indicating their status as [M−2H]2− characteristic peaks. Con-
currently, fragments that exhibit reduced intensity in the secondary mass spectrum can
Molecules 2025, 30, 2451 8 of 32

be more reliably assigned as [M−H]−. This analytical approach significantly enhances the
reliability of structural analysis for the target compound by effectively reducing the risk
of misidentification.
By using the UPLC-Q-TOF/MS instrument, (Waters Corp., Manchester, UK) the T.
chebula extract could be analyzed within 33 min, and the data were processed by KID-
NLF. As a result, a total of 164 compounds were rapidly identified (Figures 2 and 3 and
Table 1). They could be divided into three groups according to their structural types and
MS/MS fragmentation pathways.

Table 1. Characterization of chemical constituents in T. chebula by UPLC-Q-TOF/MS. (a Isolated from

T. chebula previously; b not isolated from T. chebula previously; c a newly discovered ingredient. ♠

Gallic acid derivatives; ♣ ellagitannins; ♥ triterpenoids; ♦ others. * Identified by reference standards;

# MS/MS self-built library matching. In MS/MS fragment ions, bold characters with horizontal lines
are KID, and bold characters only are NLF.)

Experi- Error
tR
No. Identification Formula mental Adducts (ppm MS/MS Fragment Ions (m/z)
(min)
(m/z) )
663.1391, 271.0435, 211.0234,
1 b,#,♠ 3-O-galloyl-glucose [17] C13H16O10 0.71 331.0662 [M−H]− −0.9
169.0134, 125.0235
2 a,*,♦ Shikimic acid [18] C7H10O5 0.75 173.0445 [M−H] −2.9
− 155.0342, 137.0235, 93.0341
711.0679, 337.0191, 293.0295,
3 a,#,♣ Neochebulic acid [19] C14H12O11 0.76 355.0298 [M−H]− −0.8
249.0401, 205.0497−
711.0682, 337.0194, 293.0297,
4 a,#,♣ Chebulic acid [20] C14H12O11 0.93 355.0303 [M−H]− 0.6
249.0402, 205.0501−
389.0355, 191.0572, 169.0141,
5 b,#,♠ 3-galloylquinic acid [21] C14H16O10 1.05 343.0661 [M−H]− −1.2
125.0240
663.1403, 271.0457 −, 211.0239,
6 a,#,♠ 1-O-galloyl-glucose [22] C13H16O10 1.17 331.0665 [M−H]− 0.0
169.0140, 125.0238
663.1408, 271.0455, 211.0244,
7 a,#,♠ 6-O-galloyl-glucose [20] C13H16O10 1.38 331.0666 [M−H]− 0.3
169.0135, 125.0238
8 a,*,♠ Gallic acid [22] C7H6O5 1.53 169.0139 [M−H]− 1.2 125.0238
663.1407, 271.0455, 211.0244,
9 a,#,♠ 2-O-galloyl-glucose [23] C13H16O10 1.55 331.0665 [M−H]− 0.0
169.0134, 125.0236
463.0544, 300.9984, 275.0194,
10 a,#,♣ Gemin D [20] C27H22O18 1.59 633.0726 [M−H]− −0.3
169.0135, 125.0234
389.0356, 191.0542, 169.0138,
11 b,#,♠ 5-galloylquinic acid [21] C14H16O10 1.72 343.0662 [M−H]− −0.9
125.0236
711.0686, 337.0196, 293.0303,
12 a,#,♣ Isochebulic acid [19] C14H12O11 1.78 355.0299 [M−H]− −0.6
249.0401, 205.0499
13 a,#,♣ Punicalin α [24] C34H22O22 1.78 781.0529 [M−H]− 0.6 600.9893, 448.9792, 300.9988
14 a,#,♣ Punicalin β [24] C34H22O22 1.85 781.0524 [M−H]− −0.4 600.9888, 448.9778, 300.9974
663.1405, 271.0455, 211.0242,
15 a,#,♠ 4-O-galloyl-glucose [23] C13H16O10 1.88 331.06663 [M−H]− −0.6
169.0132, 125.0232
Caffeic acid 3,4-O-Di glucuronide 1063.2059, 355.0307, 337.0200,
16 b,#,♦ C21H24O16 1.93 531.0993 [M−H]− 1.3
[25] 179.0710, 161.0603, 135.0446
709.0888, 687.1402, 389.0350,
17 b,#,♠ 4-galloylquinic acid [21] C14H16O10 2.19 343.0665 [M−H]− 0
191.0535, 169.0132, 125.0238
463.0517, 300.9981, 275.0190,
18 b,#,♣ Isostrictinin [26] C27H22O18 2.19 633.0728 [M−H]− 0.2
169.0136, 125.0235
541.0242, 1065.0491, 1021.0580,
19 a,#,♣ Punicacortein C [20] C48H28O30 2.33 1083.0585 [M−H]− −0.2 600.9891, 499.0722, 300.9983,
169.0136, 125.0235
Molecules 2025, 30, 2451 9 of 32

541.0250, 1065.0491, 1021.0573,

20 a,#,♣ Punicacortein D [20] C48H28O30 2.52 1083.0599 [M−H] − 1.1 600.9891, 499.0722, 300.9983,
169.0136, 125.0235
739.1009, 351.0338, 325.0563,
21 a,#,♣ 7′-O-methyl chebulate [20] C15H14O11 2.57 369.0460 [M−H]− 0.5
307.0460
651.1190, 307.0451−, 173.0440,
22 a,#,♠ 4-O-galloyl-shikimic acid [20] C14H14O9 2.74 325.0557 [M−H]− −0.9
169.0136, 155.0342, 125.0235
967.1592, 331.0670, 313.0560,
23 a,#,♠ 1,4-di-O-galloyl-β-D-glucose [23] C20H20O14 2.78 483.0778 [M−H]− 0.6 211.0242, 193.0137, 169.0135,
125.0234
691.0753, 517.0834, 499.0733,
24 a,#,♣ Chebumeinin A [27] C27H26O20 2.78 669.0943 [M−H]− 0.6 337.0200, 293.0300, 249.0397,
205.0500
651.1192, 307.0451, 173.0440,
25 a,#,♠ 5-O-galloyl-shikimic acid [20] C14H14O9 2.92 325.0559 [M−H]− −0.3
169.0136, 155.0342, 125.0235
651.1185, 307.0451−, 173.0440,
26 a,#,♠ 3-O-galloyl-shikimic acid [20] C14H14O9 3.04 325.0559 [M−H]− −0.3
169.0136, 155.0342, 125.0235
691.0773, 517.0753, 499.0724,
27 a,#,♣ Chebumeinin B [27] C27H26O20 3.21 669.0939 [M−H]− 0.0 337.0219, 293.0300, 249.0402,
205.0500
739.0991, 351.0316, 325.0558,
28 a,#,♣ 6′-O-methyl chebulate [20] C15H14O11 3.45 369.0453 [M−H]− −1.4
307.0438
939.0164, 425.0144, 407.0038,
29 a,#,♣ Valoneic acid dilactone [28] C21H10O13 3.57 469.0050 [M−H]− −0.6 300.9969, 299.9902, 169.0137,
125.0237
967.1611, 331.0666, 313.0543,
30 a,#,♠ 2,4-di-O-galloyl-β-D-glucose [29] C20H20O14 3.77 483.0780 [M−H]− 1.0 211.0245, 193.0131, 169.0134,
125.0236
541.0252, 781.0526, 600.9891,
31 a,*,♣ Punicalagin α [20] C48H28O30 3.84 1083.0583 [M−H]− −0.4
300.9983, 169.0136, 125.0235
967.1619, 331.0651, 313.0544,
32 b,#,♠ 3,4-di-O-galloyl-β-D-glucose [30] C20H20O14 3.92 483.0771 [M−H]− −0.8 271.0446, 211.0243, 193.0128,
169.0134, 125.0235
691.0770, 517.0822, 499.0734,
33 a,#,♣ Phyllanemblinin D [24] C27H26O20 4.10 669.0940 [M−H]− 0.1 337.0210, 293.0293, 249.0396,
205.0501
542.0319, 783.0687, 631.0569,
34 b,#,♣ Rhoipteleanin G [31] C48H30O30 4.39 1085.0745 [M−H]− 0.1
450.9943, 300.9985
691.0776, 499.0734, 337.0196,
35 a,#,♣ Phyllanemblinin F [20] C27H26O20 4.45 669.0939 [M−H]− 0.0
293.0295, 249.0397, 205.0500
967.1616, 331.0658, 313.0567,
36 a,#,♠ 2,6-di-O-galloyl-β-D-glucose [32] C20H20O14 4.59 483.0778 [M−H]− 0.6 271.0459, 211.0241, 193.0138,
169.0134, 125.0239
967.1622, 331.0672, 313.0565,
37 a,#,♠ 4,6-di-O-galloyl-β-D-glucose [23] C20H20O14 4.92 483.0776 [M−H]− 0.2 271.0455, 211.0243, 193.0134,
169.0134, 125.0236
463.0482, 300.9982, 275.0190,
38 a,#,♣ Strictinin [26] C27H22O18 5.08 633.0728 [M−H]− 0.0
169.0135, 125.0237
169.0134, 168.0053, 125.0230,
39 a,#,♠ Methyl gallate [20] C8H8O5 5.41 183.0290 [M−H]− −1.6
124.0160
967.1614, 331.0665, 313.0554,
40 b,#,♠ 1,2-di-O-galloyl-β-D-glucose [33] C20H20O14 5.69 483.0776 [M−H]− 0.2 271.0451, 211.0243, 193.0136,
169.0134, 125.0237
Molecules 2025, 30, 2451 10 of 32

967.1624, 331.0657, 313.0558,

41 a,#,♠ 2,3-di-O-galloyl-β-D-glucose [23] C20H20O14 6.01 483.0776 [M−H] − 0.2 271.0454, 211.0242, 193.0136,
169.0133, 125.0237
691.0761, 499.0725, 337.0203,
42 a,#,♣ Phyllanemblinin E [20] C27H26O20 6.23 669.0933 [M−H]− −0.9
293.0300, 249.0397, 205.0497
541.0252, 781.0526, 600.9891,
43 a,*,♣ Punicalagin β [20] C48H28O30 6.43 1083.0586 [M−H]− −0.1
300.9983, 169.0136
967.1631, 331.0668, 313.0558,
44 a,#,♠ 1,6-di-O-galloyl-β-D-glucose [20] C20H20O14 6.79 483.0780 [M−H]− 1.0 271.0455, 211.0242, 193.0136,
169.0134, 125.0237
967.1636, 331.0666, 313.0560,
45 a,#,♠ 3,6-di-O-galloyl-β-D-glucose [20] C20H20O14 7.21 483.0782 [M−H]− 1.4 271.0458, 211.0243, 169.0135,
125.0238
542.0331, 783.0696, 631.0578,
46 a,#,♣ Terflavin a [20] C48H30O30 7.73 1085.0756 [M−H]− 1.1
450.9946, 300.9987
47 a,#,♦ Brevifolin carboxylic acid [20] C13H8O8 7.73 291.0145 [M−H]− 1.4 337.0199, 247.0232, 203.0342
499.0485, 325.0577, 307.0452,
48 a,#,♠ 3,4-di-O-galloylshikimic acid [29] C21H18O13 8.03 477.0672 [M−H]− 0.6
169.0135, 137.0237, 125.0238
967.1615, 331.0655, 313.0557,
49 a,#,♠ 1,3-di-O-galloyl-β-D-glucose [32] C20H20O14 8.21 483.0777 [M−H]− 0.4 271.0454, 211.0240, 169.0136,
125.0237
463.0542, 300.9984, 275.0194,
50 b,#,♣ Hippomanin A [34] C27H22O18 8.35 633.0732 [M−H]− 0.6
169.0138, 125.0237
317.0391, 657.0715, 483.0771,
51 b,#,♠ 1,2,4-tri-O-galloyl-β-D-glucose [35] C27H24O18 8.57 635.0885 [M−H]− 0.2 465.0676, 313.0557, 295.0457,
169.0135, 125.0235
325.0374, 633.0739, 481.0624,
52 a,#,♣ Amlaic acid [32] C27H24O19 9.48 651.0836 [M−H]− 0.3 337.0210, 319.0083, 275.0190,
169.0135
Methyl neochebulanin [20] 705.0916, 341.0500, 513.0892,
53 a,#,♣ (4-O-methyl neochebulate-1-O-gal- C28H28O20 9.48 683.1100 [M−H]− 0.6 351.0351, 307.0457, 263.0559,
loyl-glucose) 219.0293, 204.0395,
54 b,#,♦ Phelligridin J [36] C13H6O8 9.98 288.9984 [M−H]− 1.4 245.0086
657.0726, 317.0398, 483.0783,
55 b,#,♠ 1,3,4-tri-O-galloyl-β-D-glucose [37] C27H24O18 10.19 635.0891 [M−H]− 1.1 465.0667, 313.0562, 295.0447,
169.0134, 125.0237
705.0890, 341.0493, 513.0911,
2-O-methyl neochebulate-1-O-gal-
56 c,♣ C28H28O20 10.48 683.1098 [M−H]− 0.3 351.0357, 307.0466, 263.0541,
loyl-glucose
219.0296, 204.0395
657.0710, 317.0399, 483.0784,
57 a,#,♠ 1,2,6-tri-O-galloyl-β-D-glucose [23] C27H24O18 10.90 635.0886 [M−H]− 0.3 465.0669, 313.0558, 295.0454,
169.0134, 125.0239
494.0514, 651.0831, 481.0624,
58 b,#,♣ Carpinusnin [38] C41H34O29 11.10 989.1116 [M−H]− 0.8
337.0194
807.0775, 392.0367, 615.0608,
59 a,#,♣ Tercatain [20] C34H26O22 11.56 785.0833 [M−H]− −0.5 483.0765, 463.0505, 445.0401,
300.9980
657.0705, 317.0396, 483.0775,
60 a,#,♠ 3,4,6-tri-O-galloyl-β-D-glucose [20] C27H24O18 11.69 635.0884 [M−H]− 0 465.0668, 313.0557, 295.0449,
169.0133, 125.0237
499.0492, 325.0577, 307.0466,
61 b,#,♠ 3,5-di-O-galloylshikimic acid [39] C21H18O13 11.92 477.0671 [M−H]− 0.4
169.0133, 137.0235, 125.0236
Molecules 2025, 30, 2451 11 of 32

325.0369, 633.0728, 481.0620,

62 a,#,♣ Chebulanin [20] C27H24O19 12.17 651.0833 [M−H] − −0.2 337.0202, 319.0089, 275.0195,
169.0136, 125.0236
705.0886, 341.0510, 513.0881,
6-O-methyl neochebulate-1-O-gal-
63 c,♣ C28H28O20 12.17 683.1091 [M−H]− −0.7 351.0343, 307.0459, 263.0560,
loyl-glucose
219.0291, 204.0392
485.0460, 953.0898, 935.0793,
4-galloyl-6-neochebuloyl-2,3- 801.0782, 669.0914, 633.0727,
64 c,♣ C41H32O28 12.17 971.1006 [M−H]− 0.4
HHDP-glucose 499.0726, 463.0513, 337.0202,
300.9981
499.0485, 325.0577, 307.0452,
65 b,#,♠ 4,5-di-O-galloylshikimic acid [39] C21H18O13 12.89 477.0672 [M−H]− 0.6
169.0135, 137.0237, 125.0238
705.0922, 341.0499, 513.0967,
3-O-methyl neochebulate-1-O-gal-
66 c,♣ C28H28O20 13.02 683.1088 [M−H]− −1.2 351.0341, 307.0438, 263.0558,
loyl-glucose
219.0274, 204.0385
485.0450, 953.0893, 935.0793,
1-galloyl-2-neochebuloyl-4,6- 801.0742, 669.0939, 633.0729,
67 c,♣ C41H32O28 13.29 971.1009 [M−H]− 0.7
HHDP-glucose 499.0729, 463.0508, 337.0195,
300.9981
807.0668, 392.0373, 633.0729,
68 a,#,♣ Tellimagrandin I [20] C34H26O22 13.32 785.0848 [M−H]− 1.4 483.0779, 463.0504, 445.0413,
300.9980
551.0461, 257.0085, 229.0139,
69 a,#,♦ Urolithin M5 [40] C13H8O7 13.32 275.0195 [M−H]− 1.1
201.0183
485.0457, 953.0887, 935.0786,
1-galloyl-3-neochebuloyl-4,6- 801.0779, 669.0923, 633.0719,
70 c,♣ C41H32O28 13.67 971.1014 [M−H]− 1.2
HHDP-glucose 499.0723, 463.0506, 337.0191,
300.9980
463.0514, 300.9985, 275.0189,
71 a,*,♣ Corilagin [20] C27H22O18 14.07 633.0724 [M−H]− −0.6
169.0135, 125.0235
317.0391, 657.0696, 483.0782,
72 a,#,♠ 1,4,6-tri-O-galloyl-β-D-glucose [23] C27H24O18 15.17 635.0880 [M−H]− −0.6 465.0675, 313.0558, 295.0453,
169.0134, 125.0236
317.0393, 657.0696, 483.0773,
73 b,#,♠ 2,3,4-tri-O-galloyl-β-D-glucose [41] C27H24O18 15.71 635.0881 [M−H]− −0.5 465.0668, 313.0555, 295.0451,
169.0133, 125.0238
485.0450, 953.0904, 935.0748,
1-galloyl-2-neochebuloyl-3,6- 801.0808, 669.1053, 633.0721,
74 c,♣ C41H32O28 15.96 971.1000 [M−H]− −0.2
HHDP-glucose 499.0756, 463.0502, 337.0193,
300.9983
317.0395, 657.0709, 483.0776,
75 b,#,♠ 2,4,6-tri-O-galloyl-β-D-glucose [42] C27H24O18 16.16 635.0881 [M−H]− −0.5 465.0690, 313.0558, 295.0443,
169.0134, 125.0240
807.0668, 392.0373, 633.0729,
1,3-di-O-galloyl-4,6-HHDP-glucose
76 b,#,♣ C34H26O22 16.25 785.0848 [M−H]− 1.4 483.0779, 463.0504, 445.0413,
[43]
300.9980
317.0391, 657.0693, 483.0772,
C27H24O18
77 a,#,♠ 1,3,6-tri-O-galloyl-β-D-glucose [22] 16.68 635.0872 [M−H]− −1.9 465.0667, 313.0555, 295.0449,
169.0134, 125.0234
825.0756, 401.0424, 785.0836,
1,3-di-O-galloyl-2,4-chebuloyl-D-
78 a,#,♣ C34H28O23 17.10 803.0942 [M−H]− −0.1 633.0723, 589.0815,533.0569,
glucose [44]
483.0198, 313.0563
Molecules 2025, 30, 2451 12 of 32

485.0457, 953.0826, 935.0793,

Neochebulagic acid [20]
801.0682, 669.1024, 633.0735,
79 a,#,♣ (1-galloyl-4-neochebuloyl-3,6- C41H32O28 17.57 971.0999 [M−H]− −0.3
499.0777, 463.0483, 337.0194,
HHDP-glucose)
300.9984
809.0811, 393.0450, 635.0889,
1,2,3,6-tetra-O-galloyl-β-D-glucose
80 a,#,♠ C34H28O22 17.62 787.0999 [M−H]− 0.6 617.0783, 483.0782, 465.0676,
[20]
447.0562, 313.0555, 295.0450
468.0425, 767.0801, 635.0894,
81 a,#,♣ Tellimagrandin II [18] C41H30O26 17.82 937.0959 [M−H]− 1.3
465.0661, 313.0561, 300.9982
317.0401, 657.0699, 483.0765,
82 b,#,♠ 2,3,6-tri-O-galloyl-β-D-glucose [35] C27H24O18 17.97 635.0879 [M−H]− −0.8 465.0678, 313.0568, 295.0454,
169.0136, 125.0237
486.0532, 803.0942, 635.0881,
1,2,3-tri-O-galloyl-4-neochebuloyl-
83 c,♣ C41H34O28 18.15 973.1159 [M−H]− 0.1 633.0729, 483.0779, 465.0667,
D-glucose
463.0511, 337.0193, 300.9985
541.0252, 781.0580, 600.9910,
84 a,#,♣ Terchebulin [45] C48H28O30 19.00 1083.0604 [M−H]− 1.6 448.9790, 300.9985, 169.0136,
125.0236
657.0691, 317.0393, 483.0773,
85 a,#,♠ 1,2,3-tri-O-galloyl-β-D-glucose [23] C27H24O18 19.24 635.0880 [M−H]− −0.6 465.0668, 313.0558, 295.0443,
169.0133, 125.0238
476.0416, 783.0739, 651.0840,
1-O-galloyl-3,4-chebuloyl-2,6- 633.0735, 481.0611, 463.0504,
86 c,♣ C41H30O27 19.65 953.0919 [M−H]− 2.4
HHDP-D-glucose 337.0199, 331.0665,
319.0095, 300.9988
485.0459, 953.0898, 935.0773,
1-galloyl-4-neochebuloyl-2,3- 801.0789, 669.0975, 633.0717,
87 c,♣ C41H32O28 19.92 971.1003 [M−H]− 0.1
HHDP-glucose 499.0704, 463.0513, 337.0197,
300.9984
283.9958, 257.0087, 229.0122,
88 a,*,♣ Ellagic acid [20] C14H6O8 20.04 300.9987 [M−H]− 1.0 201.0182, 185.0236, 173.0223,
145.0288, 117.0326
1-O-galloyl-3,4-THDP-2,6-HHDP- 462.0426, 773.0887, 755.0728,
89 c,♣ C40H30O26 20.13 925.0954 [M−H]− 0.8
D-glucose 633.0735, 463.0518, 300.9986
476.0406, 783.0743, 651.0848,
1-O-galloyl-3,6-chebuloyl-2,4- 633.0744, 481.0629, 463.0519,
90 c,♣ C41H30O27 20.27 953.0895 [M−H]− −0.1
HHDP-D-glucose 337.0198, 331.0643,
319.0092, 300.9991
468.0435,767.0743, 635.0884,
91 b,#,♣ Punicafolin [38] C41H30O26 20.29 937.0961 [M−H]− 1.5
465.0688, 313.0545, 300.9987
401.0431, 825.0756, 785,0836,
1,6-di-O-galloyl-2,4-chebuloyl-D-
92 c,♣ C34H28O23 20.37 803.0957 [M−H]− 1.7 633.0723, 589.0815, 533.0569,
glucose
483.0198, 313.0563
462.0449, 773.0849, 755.0762,
93 b,#,♣ Phyllantusiin C [30] C40H30O26 20.37 925.0955 [M−H]− −0.3
633.0731, 463.0514, 300.9985
486.0536, 803.0956, 635.0879,
1,3,6-tri-O-galloyl-4-neochebuloyl-
94 a,♣ C41H34O28 20.50 973.1162 [M−H]− 0.4 633.0724, 483.0755, 465.0685,
D-glucose [29]
463.0514, 337.0192, 300.9984
476.0412, 783.0712, 651.0827,
1-O-galloyl-4,6-chebuloyl-2,3- 633.0726, 481.0653, 463.0526,
95 c,♣ C41H30O27 20.60 953.0893 [M−H]− −0.3
HHDP-D-glucose 337.0191, 331.0658,
319.0093, 300.9987
Molecules 2025, 30, 2451 13 of 32

1-O-galloyl-3,6-THDP-2,4-HHDP- 462.0430, 773.0862, 755.0744,

96 c,♣ C40H30O26 20.77 925.0959 [M−H]− 1.3
D-glucose 633.0735, 463.0521, 300.9988
468.0435, 767.0731, 635.0885,
97 b,#,♣ Davidiin [46] C41H30O26 20.86 937.0964 [M−H]− 1.8
465.0673, 313.0563, 300.9987
825.0776, 401.0432, 785.0864,
3,6-di-O-galloyl-2,4-chebuloyl-D-
98 c,♣ C34H28O23 20.90 803.0961 [M−H]− 2.2 633.0743, 589.0851, 533.0582,
glucose
483.0163, 313.0558
477.0655, 459.0561, 325.0501,
99 c,♠ 3,4,5-tri-O-galloyl shikimic acid C28H22O18 20.90 629.0789 [M−H]− 1.6
307.0443, 289.0357, 169.0138
486.0546, 803.0958, 635.0894,
1,2,6-tri-O-galloyl-4-neochebuloyl-
100 c,♣ C41H34O28 20.94 973.1168 [M−H]− 1.0 633.0729, 483.0772, 465.0677,
D-glucose
463.0529, 337.0198, 300.9986
485.0463, 953.0900, 935.0801,
2-galloyl-3-neochebuloyl-4,6- 801.0813, 669.0943, 633.0731,
101 c,♣ C41H32O28 20.94 971.1013 [M−H]− 1.1
HHDP-glucose 499.0749, 463.0515, 337.0198,
300.9986
102 a,#,♣ Eschweilenol C [20] C20H16O12 21.01 447.0573 [M−H]− 2.0 895.1224, 300.9984
1-O-galloyl-4,6-THDP-2,3-HHDP- 462.0424, 773.0867, 755.0684,
103 c,♣ C40H30O26 21.07 925.0956 [M−H]− 1.0
D-glucose 633.0731, 463.0518, 300.9983
1,3,4-tri-O-galloyl-2,6-HHDP-glu- 468.0437, 767.0753, 635.0886,
104 c,♣ C41H30O26 21.10 937.0964 [M−H]− 1.8
cose 465.0679, 313.0557, 300.9991
476.0415, 783.0693, 651.0840,
633.0735, 481.0627, 463.0518,
105 a,*,♣ Chebulagic acid [22] C41H30O27 21.15 953.0896 [M−H]− 1.7
337.0198, 331.0667,
319.0092, 300.9990
486.0541, 803.0955, 635.0891,
2,3,6-tri-O-galloyl-4-neochebuloyl-
106 c,♣ C41H34O28 21.29 973.1172 [M−H]− 1.4 633.07235, 483.0763, 465.0677,
D-glucose
463.0523, 337.0198, 300.9992
468.0432, 767.0742, 635.0887,
107 b,#,♣ Pterocarinin C [47] C41H30O26 21.34 937.0959 [M−H]− 1.3
465.0670, 313.0559, 300.9989
809.0834, 393.0457, 635.0889,
1,2,3,4-tetra-O-galloyl-β-D-glucose
108 a,#,♠ C34H28O22 21.35 787.0999 [M−H]− 0.6 617.0782, 483.0770, 465.0676,
[23]
447.0555, 313.0556, 295.0450
485.0464, 953.0909, 935.0782,
1-galloyl-6-neochebuloyl-2,3- 801.0809, 669.1044, 633.0738,
109 c,♣ C41H32O28 21.39 971.1014 [M−H]− 1.2
HHDP-glucose 499.0724, 463.0503, 337.0197,
300.9990
807.0668, 392.0373, 633.0729,
1,6-di-O-galloyl-2,3-HHDP-glucose
110 b,#,♣ C34H26O22 21.47 785.0848 [M−H]− 1.4 483.0779, 463.0504, 445.0413,
[48]
300.9980
1,2,3-tri-O-galloyl-4-methyl neo- 493.0620, 817.1097, 635.0881,
111 c,♣ C42H36O28 21.74 987.1318 [M−H]− 0.3
chebuloyl-glucose 465.0668, 351.0352, 295.0450
809.0809, 393.0447, 635.0881,
1,2,4,6-tetra-O-galloyl-β-D-glucose
112 a,#,♠ C34H28O22 21.85 787.0994 [M−H]− 0.0 617.0773, 483.0768, 465.0674,
[23]
447.0560, 313.0555, 295.0451
492.0533, 815.0938, 683.0846,
1-O-galloyl-3,6-HHDP-4-6′ methyl
113 a,#,♣ C42H34O28 21.94 985.1156 [M−H]− −0.2 633.0719, 513.0870, 463.0513,
neochebuloyl-glucose [49]
351.0342, 300.9983
809.0819, 393.0447, 635.0874,
1,3,4,6-tetra-O-galloyl-β-D-glucose
114 a,#,♠ C34H28O22 22.00 787.0995 [M−H]− 0.1 617.0775, 483.0771, 465.0668,
[20]
447.0558, 313.0556, 295.0449
Molecules 2025, 30, 2451 14 of 32

492.0534, 815.0939, 683.0846,

c,♣
2-O-galloyl-3,6-HHDP-4-6′ methyl
115 C42H34O28 22.13 985.1154 [M−H]− −0.4 633.0718, 513.0867, 463.0512,
neochebuloyl-glucose
351.0346, 300.9983
1-O-galloyl-2,3-THDP-4,6-HHDP- 462.0423, 773.0825, 755.0762,
116 c,♣ C40H30O26 22.20 925.0941 [M−H]− −0.6
D-glucose 633.0716, 463.0508, 300.9982
807.0775, 392.0367, 615.0608,
1,2-di-O-galloyl-4,6-HHDP-glucose
117 a,#,♣ C34H26O22 22.29 785.0833 [M−H]− −0.5 483.0765, 463.0505, 445.0401,
[50]
300.9980
485.0472, 953.0893, 935.0806,
2-galloyl-4-neochebuloyl-3,6- 801.0945, 669.0826, 633.0731,
118 c,♣ C41H32O28 22.49 971.1006 [M−H]− 0.4
HHDP-glucose 499.0716, 463.0505, 337.0193,
300.9982
476.0383, 783.0655, 651.0851,
1-O-galloyl-2,6-chebuloyl-3,4- 633.0722, 481.0618, 463.0501,
119 c,♣ C41H30O27 22.65 953.0883 [M−H]− −1.4
HHDP-D-glucose 337.0194, 331.0634,
319.0079, 300.9979
809.0819, 393.0447, 635.0871,
2,3,4,6-tetra-O-galloyl-β-D-glucose
120 b,#,♠ C34H28O22 22.66 787.0986 [M−H]− −1.0 617.0767, 483.0764, 465.0662,
[51]
447.0559, 313.0554, 295.0446
485.0458, 953.0894, 935.0800,
6-galloyl-4-neochebuloyl-2,3- 801.0879, 669.0791, 633.0726,
121 c,♣ C41H32O28 22.84 971.0999 [M−H]− −0.3
HHDP-glucose 499.0721, 463.0498, 337.0190,
300.9981
1,2,3-tri-O-galloyl-4,6-neo- 477.0480, 785.0828, 617.0756,
122 c,♣ C41H32O27 23.03 955.1048 [M−H]− −0.5
chebuloyl-glucose 465.0661, 447.0555, 337.0194
476.0395, 783.0699, 651.0839,
1-O-galloyl-2,3-chebuloyl-4,6- 633.0712, 481.0641, 463.0492,
123 c,♣ C41H30O27 23.76 953.0882 [M−H]− −1.5
HHDP-D-glucose 337.0190, 331.0685,
319.0087, 300.9981
1,2,4-tri-O-galloyl-3,6-neo- 477.0481, 785.0828, 617.0765,
124 c,♣ C41H32O27 23.82 955.1045 [M-H]− −0.8
chebuloyl-glucose 465.0669, 447.0562, 337.0189
1,3,6-tri-O-galloyl-4-methyl neo- 493.0611, 817.1092, 635.0872,
125 a,#,♣ C42H36O28 23.93 987.1302 [M−H]− 1.1
chebuloyl-glucose [20] 465.0662, 351.0343, 295.0445
1,2,6-tri-O-galloyl-4-methyl neo- 493.5631, 817.1094, 635.0876,
126 c,♣ C42H36O28 24.36 987.1313 [M−H]− −0.2
chebuloyl-glucose 465.0673, 351.0338, 295.0440
Chebulinic acid [20] 477.0486, 785.0839, 635.0873,
127 a,*,♣ (1,3,6-tri-O-galloyl-2,4-chebuloyl- C41H32O27 24.57 955.1054 [M−H]− 0.1 617.0771, 465.0668, 447.0558,
glucose) 337.0190
2,3,6-tri-O-galloyl-4-methyl neo- 493.0623, 817.1107, 635.0883,
128 c,♣ C42H36O28 25.47 987.1324 [M−H]− 0.9
chebuloyl-glucose 465.0674, 351.0348, 295.0458
1,4,6-tri-O-galloyl-2,3-neo- 477.0491, 785.0850, 617.0781,
129 c,♣ C41H32O27 25.47 955.1041 [M−H]− −1.3
chebuloyl-glucose 465.0674, 447.0564, 337.0197
469.0515, 787.1005, 769.0900,
617.0784, 599.0676, 465.0677,
1,2,3,4,6-penta-O-galloyl-β-D-glu-
130 a,*,♠ C41H32O26 26.73 939.1113 [M−H]− 1.0 447.0568, 429.0460, 313.0565,
cose [22]
295.0456, 277.0352, 259.0245,
169.0136, 125.0237
935.0799, 917.0695, 617.0781,
131 a,#,♣ Terchebin [52] C41H30O27 28.19 953.0901 [M−H]− 0.5
465.0671, 316.9932, 295.0452
4-O-(4″-O-galloyl-α-rhamnopyra- 1199.1431, 621.0486, 447.0560,
132 a,#,♣ C27H20O16 28.94 599.0673 [M−H]− 0.0
nosyl) ellagic acid [49] 429.0441, 300.9985
1-O-galloyl-2,6-THDP-3,4-HHDP- 462.0432, 773.0815, 755.0752,
133 c,♣ C40H30O26 29.04 925.0959 [M−H]− 1.3
D-glucose 633.0731, 463.0506, 300.9982
Molecules 2025, 30, 2451 15 of 32

4-O-(2″,3″-di-O-galloyl-α-L-rham- 375.0342, 599.0668, 581.0553,

134 a,#,♣ C34H24O20 29.31 751.0782 [M−H]− −0.1
nosyl) ellagic acid [20] 449.0718, 411.0366, 300.9985
468.0433, 767.0732, 635.0894,
135 b,#,♣ Nupharin A [53] C41H30O26 29.35 937.0955 [M−H]− 0.9
465.0674, 313.0573, 300.9988
136 c,♠ 1-O-cinnamoyl-6-O-galloyl-glucose C22H22O11 29.39 461.1084 [M−H]− 0 313.0558, 169.0135, 147.0436
4-O-(2″,4″-di-O-galloyl-α-L-rham- 375.0342, 599.0668, 581.0553,
137 a,#,♣ C34H24O20 29.42 751.0782 [M−H]− −0.1
nosyl) ellagic acid [20] 449.0718, 411.0366, 300.9985
4-O-(3″,4″-di-O-galloyl-α-rhamno- 375.0338, 599.0660, 581.0549,
138 a,#,♣ C34H24O20 29.50 751.0776 [M−H]− −0.9
pyranosyl) ellagic acid [49] 449.0713, 411.0292, 300.9977
Arjungenin-24-O-glucoheptonic
139 c,♥ C37H60O13 29.59 711.3952 [M−H]− −0.6 503.3379
acid
1-O-galloyl-2-O-cinnamoyl-glucose 923.2247, 313.0561, 169.0133,
140 b,#,♠ C22H22O11 29.73 461.1092 [M−H]− 1.7
[54] 147.0443
141 a,#,♥ Quercotriterpenoside I [55] C43H62O15 29.73 817.4018 [M−H]− 1.0 655.3484, 503.3366
1-O-galloyl-6-O-cinnamoyl-glucose
142 b,#,♠ C22H22O11 29.81 461.1077 [M−H]− −1.5 313.0562, 169.0132, 147.0443
[54]
1,2-O-galloyl-6-O-cinnamoyl-glu- 465.0682, 461.1077, 313.0560,
143 a,#,♠ C29H26O15 29.82 613.1190 [M−H]− −0.5
cose [20] 169.0131, 147.0443
1-O-cinnamoyl-2-O-galloyl-glucose
144 b,#,♠ C22H22O11 29.95 461.10978 [M−H]− −1.3 313.0549, 169.0132, 147.0434
[56]
Madecassic acid-24-galloyl-28-glu-
145 c,♥ C43H62O15 30.13 817.4008 [M−H]− −0.2 655.3470, 503.3369
cose
1-O-cinnamoyl-2,6-O-galloyl-glu- 465.0667, 461.1085, 313.0558,
146 b,#,♠ C29H26O15 30.19 613.1190 [M−H]− −0.5
cose [57] 169.0131, 147.0441
Terminolic acid-24-galloyl-28-glu-
147 a,#,♥ C43H62O15 30.27 817.3998 [M−H]− −1.5 655.3473, 503.3361
cose [56]
Rotundic acid-24-galloyl-28-glu-
148 c,♥ C43H62O14 30.30 801.4066 [M−H]− 0.6 639.3536, 487.3401
cose
Madecassic acid-24-O-glucohep-
149 c,♥ C37H60O13 30.34 711.3954 [M−H]− −0.3 503.3372
tonic acid
Rotundic acid-24-O-glucoheptonic
150 c,♥ C37H60O12 30.37 695.3983 [M−H]− −3.5 487.3413
acid
151 c,♥ Asiatic acid-24-galloyl-28-glucose C43H62O14 30.39 801.4073 [M−H]− 1.5 639.3538, 487.3424
1,6-O-galloyl-2-O-cinnamoyl-glu- 465.0645, 461.1104, 313.0572,
152 a,#,♠ C29H26O15 30.39 613.1200 [M−H]− 1.1
cose [20] 169.0134, 147.0451
Terminolic acid-24-O-glucohep-
153 c,♥ C37H60O13 30.43 711.3954 [M−H]− −0.3 503.3377
tonic acid
Asiatic acid-24-O-glucoheptonic C37H60O1
154 c,♥ 30.47 695.4011 [M−H]− 0.6 487.3428
acid 2

Arjunolic acid-24-galloyl-28-glu-
155 c,♥ C43H62O14 30.51 801.4060 [M−H]− 1–0.1 639.3539, 487.3427
cose
Arjunolic acid-24-O-glucoheptonic
156 c,♥ C37H60O12 30.57 695.4019 [M−H]− 0.6 487.3432
acid
811.4279, 787.1131, 635.0932,
a,#,♠
1,2,3-tri-O-galloyl-6-O-cinnamoyl-
157 C36H30O19 30.57 765.1310 [M−H]− 0.9 617.0841, 613.1192, 595.1093,
β- D-glucose [20]
443.0975
158 a,#,♥ Arjungenin [40] C30H48O6 31.12 503.3384 [M−H]− 2.2 1007.6838, 549.3438
159 a,#,♥ Madecassic acid [28] C30H48O6 31.56 503.3387 [M−H]− 2.8 1007.6841, 549.3439
160 a,#,♥ 23-galloyl-arjunolic acid [58] C37H52O9 31.71 639.3547 [M−H]− 2.2 487.3378
161 a,#,♥ Terminolic acid [55] C30H48O6 31.77 503.3387 [M−H]− 2.8 1007.6847, 549.3439
162 b,#,♥ Rotundic acid [59] C30H48O5 31.90 487.3437 [M−H]− 2.9 975.6937
163 a,#,♥ Asiatic acid [32] C30H48O5 32.09 487.3438 [M−H]− 3.1 975.6949
164 a,#,♥ Arjunolic acid [18] C30H48O5 32.35 487.3434 [M−H]− 2.3 975.6979
Molecules 2025, 30, 2451 16 of 32

2.3.1. Gallic Acid Derivatives

In T. chebula, the primary compounds of gallic acid and its derivatives consist of sim-
ple gallic acid components and their acyl esters. The main constituents identified are gallic
acid (8) and methyl gallate (39). These compounds are characterized by multiple phenolic
hydroxyl and carboxyl groups, which render them susceptible to the loss of molecules
such as CO2, CO, and H2O during cleavage. A relatively high content of gallic acid is pre-
sent in myrobalan, with two significant ion peaks observed at m/z 169 and m/z 125 in the
primary mass spectrum. Based on the peak elution time and ionic strength of the com-
pounds, m/z 169 has been confirmed as the deprotonated molecular ion [M−H]−, while m/z
125 is identified as the main fragment ion resulting from in-source fragmentation, with
the molecular formula C7H6O5. Further analysis indicates that the primary secondary frag-
ment is the ion at m/z 125.0238 [M−H−CO2]−, formed by the removal of a carboxyl group
(CO2, Δm = 44). By comparing with the mass spectrometry data of the reference substance,
peak 8 has been confirmed as gallic acid. The primary mass spectrum of peak 39 exhibits
a prominent abundance at m/z 183, which is identified as a deprotonated molecular ion
[M−H]−, corresponding to the molecular formula C8H8O5. In the secondary mass spectrum,
in addition to the m/z 169 and m/z 125 fragments of gallic acid, characteristic fragments
m/z 168 [M−H−CH3]− and m/z 124.0160 [M−H−CH3−CO2]− have also been detected. Conse-
quently, peak 39 has been confirmed as methyl gallate.
Simple galloyl ester compounds are mainly divided into three categories: gallic acid
bound to glucose (Glc), gallic acid bound to quinic acid, and gallic acid bound to shikimic
acid. In this study, 38 gallotannin components were identified. These components were
classified according to the number of galloyl groups attached to the Glc molecules. The
categories include mono-galloyl glucose (1, 6, 7, 9, 15), di-galloyl glucose (23, 30, 32, 36,
37, 40, 41, 44, 45, 49), tri-galloyl glucose (51, 55, 57, 60, 72, 73, 75, 77, 82, 85), tetra-galloyl
glucose (80, 108, 112, 114, 120), and penta-galloyl glucose (130). Characteristic neutral
losses were observed as galloyl and gallic acid moieties were gradually removed (Figure
5).
Molecules 2025, 30, 2451 17 of 32

Figure 5. Schematic putative fragmentation pattern of gallotannins. The positions of galloyl groups
on the sugar structure are indicated by numbers in circular shapes of different colors. The number
of galloyl groups in glycoside is shown by red dashed boxes.

The mass spectrum of penta-galloyl glucose (m/z 939) demonstrated a sequential loss
of galloyl groups, resulting in the formation of tetra-galloyl glucose (m/z 787), tri-galloyl
glucose (m/z 635), di-galloyl glucose (m/z 483), and mono-galloyl glucose (m/z 331). Addi-
tionally, the primary intermediate ions observed for di-galloyl glucose and mono-galloyl
glucose were m/z 271 and m/z 211, respectively. These fragment ions were attributed to
the continuous loss of -CHOH groups from the glucose moiety, indicating that mono-
galloyl glucose undergoes fragmentation to yield [M−H−60]− and [M−H−60−60]−.
Based on the number and structure of galloyl and cinnamoyl groups, a series of iso-
mers can be distinguished, and their peaks can be clearly identified. These compounds
are categorized as cinnamoyl-mono-galloyl glucose (136, 140, 142, 144), cinnamoyl-di-gal-
loyl glucose (143, 146, 152), and cinnamoyl-tri-galloyl glucose (157). In the mass spectrum,
cinnamoyl-tri-galloyl glucose (m/z 765) exhibits a gradual loss of the gallic acid compo-
nent, leading to the formation of cinnamoyl-di-galloyl glucose (m/z 595), cinnamoyl-
mono-galloyl glucose (m/z 425), and cinnamoyl glucose (m/z 255) (Figure 6). Additionally,
characteristic diagnostic ions of m/z 169, attributed to gallic acid, are observed in the mass
spectrometry analysis. Ion fragments of m/z 125 are generated through the neutral loss of
carboxyl groups (CO2, Δm = 44), while m/z 103 is formed by the loss of carboxyl groups
(CO2, Δm = 44) from cinnamic acid, serving as a distinctive fragment for cinnamic acid.
Molecules 2025, 30, 2451 18 of 32

For the position isomers, the calculated lipophilicity parameter (ClogP) was used to esti-
mate the retention time of isomers in the reversed-phase column as the basis for differen-
tiation. Generally, compounds with a larger ClogP value would retain longer. The struc-
tures of these isomers were ultimately assigned by combining peak times with calculated
ClogP values (Table S1).

Figure 6. Schematic putative fragmentation pattern of galloyl derivatives of cinnamic acid. The po-
sitions of galloyl groups on the sugar structure are indicated by numbers in circular shapes of dif-
ferent colors. The number of galloyl groups in glycoside is shown by red dashed boxes.

Another type of simple galloyl ester is formed by the combination of gallic acid and
shikimic acid. Based on the number of galloyl groups, these compounds can be catego-
rized as mono-galloyl shikimic acid, di-galloyl shikimic acid, and tri-galloyl shikimic acid.
In this study, a total of seven related compounds were identified and classified according
to the number of galloyl groups connected to shikimic acid, including mono-galloyl shi-
kimic acid (22, 25, 26), di-galloyl shikimic acid (48, 61, 65), and tri-galloyl shikimic acid
(99). Prominent characteristic fragment ions were observed in the mass spectrum through
the sequential elimination of galloyl and shikimic acid moieties. Tri-galloyl shikimic acid
(m/z 629) demonstrated continuous mass loss of the galloyl moiety, leading to the for-
mation of di-galloyl shikimic acid (m/z 477) and mono-galloyl shikimic acid (m/z 325). A
fragment ion of m/z 169 was detected for all components and identified as [M−H]− of gallic
acid, resulting in a fragment of m/z 125 through the neutral loss of a carboxyl group (CO2,
Δm = 44). In addition, fragment ions of m/z 155 and m/z 137 were attributed to the loss of
water molecules (H2O, Δm = 18) from shikimic acid. Similarly, further removal of the car-
boxyl group (CO2, Δm = 44) after water loss resulted in fragment ions of m/z 111 and m/z
Molecules 2025, 30, 2451 19 of 32

93, which are considered characteristic fragments of shikimic acid. Finally, by analyzing
the arrangement of galloyl groups at different positions on shikimic acid, a series of iso-
mers was identified (Figure 7). The assignment of each peak was determined by examin-
ing peak times and ClogP values (Table S1).

Figure 7. Schematic putative fragmentation pattern of galloyl derivatives of shikimic acid. The po-
sitions of galloyl groups on the shikimic acid structure are indicated by numbers in circular shapes
of different colors. The number of galloyl groups in shikimic acid is shown by red dashed boxes.

The final type of simple galloyl ester is formed by the combination of gallic acid and
quinic acid. This compound typically produces a characteristic fragment at m/z 191. While
components with two or three galloyl groups combined with quinic acid may exist, none
were identified in this study. A total of three isomers of mono-galloyl quinic acid were
identified. In the primary mass spectrum, peaks 5, 11, and 17 all displayed ion peaks at
m/z 389 and m/z 343. Notably, the intensity of peak 17 was significantly higher than that
of peaks 5 and 11, approximately five times greater. Additionally, peak 17 presented frag-
ments at m/z 709 and m/z 687. In the secondary mass spectrum, the ion peak intensities of
m/z 709, m/z 687, and m/z 389 were all reduced. Based on these observations, it was con-
cluded that m/z 709 corresponds to the [2M+Na−2H]⁻ ion peak, m/z 687 corresponds to the
[2M−H]⁻ ion peak, and m/z 389 represents the adduct ion [M+HCOO]⁻, with the
Molecules 2025, 30, 2451 20 of 32

corresponding molecular formula being C16H14O10. Fragment ion peaks of m/z 191 [quinic
acid−H]⁻ and m/z 169 [gallic acid−H]⁻ can be generated through the neutral loss of quinic
acid or gallic acid. By incorporating relevant literature, the order of the peaks was further
clarified, leading to the identification of peak 5 as 3-galloyl quinic acid, peak 11 as 5-galloyl
quinic acid, and peak 17 as 4-galloyl quinic acid.

2.3.2. Ellagitannins
Ellagitannins are a significant class of polyphenolic compounds containing one or
more hexahydroxydiphenoyl (HHDP) groups or their oxidized forms, such as dehydro-
hexahydroxydiphenoyl (DHHDP) and chebuloyl (Che). Upon hydrolysis, they yield sta-
ble ellagic acid. In negative ion mode, ellagic acid tannins exhibit characteristic fragment
ion losses, including HHDP (302 Da), DHHDP (318 Da), and chebuloyl (320 Da) (Figure
4).

Ellagic Acid and Its Simple Derivatives

The simple derivatives of ellagic acid primarily consist of ellagic acid conjugated with
rhamnose to form glycosides at the 1-position, with additional hydroxyl groups on the
rhamnose linked to multiple galloyl groups. MS analysis reveals that peaks 134, 137, and
138 display identical ions at m/z 375 and m/z 751 in the primary MS. The disappearance of
m/z 375 ions in the secondary MS suggests that m/z 375 corresponds to the [M−2H]2⁻ ion
peak. The m/z 751 ion is identified as the [M−H]⁻ ion peak, corresponding to the molecular
formula C34H24O20. In the secondary mass spectrum, fragment ions were observed at m/z
599 [M−H−152]⁻, m/z 581 [M−H−170]⁻, m/z 449 [M−H−302]⁻, m/z 411 [M−H−170−170]⁻, and
m/z 301 [M−H−152−152−146]⁻/[ellagic acid−H]⁻, generated by the sequential loss of galloyl,
gallic, and ellagic acid groups. The polarity was assessed using ClogP values (Table S1),
leading to the identification of peak 134 as 4-O-(2″,3″-di-O-galloyl-α-L-rhamnosyl) ellagic
acid, peak 137 as 4-O-(2″,4″-di-O-galloyl-α-L-rhamnosyl) ellagic acid and peak 138 as 4-
O-(3″,4″-di-O-galloyl-α-rhamnopyranosyl) ellagic acid. Similar cleavage patterns were ob-
served for peaks 102 and 132, where peak 102 was identified as Eschweilenol C and peak
132 as 4-O-(4″-O-galloyl-α-rhamnopyranosyl) ellagic acid. Additionally, ellagic acid can
directly conjugate with gallic acid. In the primary mass spectrum of peak 29, ion peaks
were observed at m/z 469 and m/z 939. The intensity of the m/z 939 ion peak diminished in
the secondary mass spectrum, indicating that m/z 939 corresponds to the [2M−H]⁻ ion
peak, while m/z 469 corresponds to the [M−H]⁻ ion peak, with the molecular formula
C21H10O13. Fragment ion peaks at m/z 425 [M−H−CO2]⁻, m/z 407 [M−H−CO2−H2O]⁻, and m/z
299 [M−H−gallic acid]⁻ were generated by the loss of CO2 and H2O. Peak 29 was identified
as Valoneic acid dilactone. Terminalia chebula contains a significant amount of free ellagic
acid. Numerous studies have focused on the quantification of ellagic acid using
HPLC/UPLC. Peak 88 was identified as ellagic acid through comparison with reference
standards.

Simple Tannins Containing a Single HHDP Group

Simple tannins containing a single HHDP group are primarily composed of com-
pounds with one HHDP group and may include one or more attached galloyl groups.
Based on the number of galloyl groups, these compounds can be classified into three
types: mono-galloyl-HHDP glucose (10, 18, 38, 50, 71), di-galloyl-HHDP glucose (59, 68,
76, 110, 117), and tri-galloyl-HHDP glucose (81, 91, 97, 104, 107, 135). In mass spectrome-
try, these compounds exhibit a characteristic cleavage pattern, initially losing the HHDP
group, followed by sequential losses of galloyl groups. The mass spectrum shows a char-
acteristic neutral loss of 302 Da, indicative of HHDP group cleavage. For instance, tri-
galloyl-HHDP glucose (m/z 937) first loses the HHDP group to generate tri-galloyl glucose
Molecules 2025, 30, 2451 21 of 32

(m/z 635), which then fragments following the cleavage pathway typical for tri-galloyl
glucose. Similarly, mono-galloyl-HHDP glucose and di-galloyl-HHDP glucose exhibit
comparable fragmentation patterns. Additionally, this class of compounds also produces
a characteristic fragment at m/z 275, primarily generated by the elimination of the HHDP
group, followed by the loss of a galloyl group and subsequently glucose.

Chebulic Acid and Its Simple Derivatives

The primary chebulic acids found in Terminalia chebula are neochebulic acid (3),
chebulic acid (4), and isochebulic acid (12). These compounds are rich in phenolic hy-
droxyl and carboxyl groups, making them prone to losing CO2, H2O, and other groups
during mass spectrometry fragmentation. In the primary mass spectrum, ion peaks at m/z
711, m/z 355, and m/z 337 correspond to peaks 3, 4, and 12, respectively. These ion peaks
are also observed in the secondary mass spectrum. The m/z 711 peak is presumed to be
the [2M−H]⁻ ion, m/z 355 as the [M−H]⁻ ion, and m/z 337 as a fragment with the molecular
formula C14H12O11. During pyrolysis, the sequential loss of H2O and CO2 generates frag-
ment ion peaks at m/z 293 [M−H−H2O−CO2]⁻, m/z 249 [M−H−H2O−2CO2]⁻, and m/z 205
[M−H−H2O−3CO2]⁻. The polarity was assessed using ClogP values (Table S1), and peak
order was established by comparison with reference standards. Consequently, peak 3 was
identified as isochebulidic acid, peak 4 as chebulidic acid, and peak 12 as neochebulidic
acid. In the primary mass spectrum, peaks 21 and 28 exhibit ion peaks at m/z 369 and m/z
739, respectively. In the secondary mass spectrum, the intensity of the m/z 739 ion peak
diminishes, suggesting that m/z 739 corresponds to a [2M−H]⁻ ion, while m/z 369 corre-
sponds to a [M−H]⁻ ion, with the molecular formula C15H14O11. During pyrolysis, these
compounds produce fragment ion peaks at m/z 351 [M−H−H2O]⁻, m/z 325 [M−H−CO2]⁻,
and m/z 307 [M−H−H2O−CO2]⁻ through the sequential loss of H2O and CO2. Peak 21 was
identified as 7′-O-methylchebulate and peak 28 as 6′-O-methylchebulate.

Simple Tannins Containing a Single Chebuloyl or Neoche Group

In this study, the primary types of simple tannin-like components within the
chebuloyl, neoche, and methylneoche groups were identified through mass spectrometry.
These components primarily consist of a chebuloyl (320 Da), neoche (338 Da), or methyl-
neoche (352 Da) group and their related derivatives, potentially containing multiple gal-
loyl groups. The molecular weight difference between the chebuloyl and neoche groups
is equivalent to one H2O molecule (18 Da). The key distinction between these groups lies
in the attachment: the chebuloyl group is linked to two glycohydroxyl groups, while the
neoche group is linked to only one. Based on the number of galloyl groups, these com-
pounds can be further categorized as mono galloyl-neoche glucose (24, 27, 33, 35, 42, 52,
53, 56, 62, 63, 66), bisgalloyl-chebuloyl glucose (78, 92, 98), and trigaloyl-chebuloyl/neoche
glucose (83, 94, 100, 106, 111, 122, 124, 125, 126, 127, 128, 129). These compounds exhibited
neutral losses of 320 Da, 338 Da, or 352 Da in the mass spectrum. This indicates that these
compounds are prone to losing chebuloyl, neoche, or methylneoche groups during mass
spectrometric analysis. Specifically, the fragmentation of trigaloyl-chebuloyl/neoche glu-
cose results in the loss of the chebuloyl/neoche group, forming trigaloyl glucose (m/z 635),
which then undergoes further cleavage following the pattern typical of trigaloyl glucose.
Likewise, monogalloyl-chebuloyl/neoche glucose displayed a comparable fragmentation
pattern.

Tannins Containing HHDP, Chebuloyl, Neoche, and Other Groups

Terminalia chebula contains various tannins, which typically include groups such as
HHDP (302 Da), chebuloyl (320 Da), neoche (338 Da), THDP (292 Da), DHHDP (318 Da),
flavogallonyl (452 Da), and Gallagyl (602 Da). The combinations of these groups are
Molecules 2025, 30, 2451 22 of 32

complex and varied, potentially including HHDP along with other groups such as
chebuloyl, neoche, THDP, DHHDP, Gallagyl, or flavogallonyl. Different components can
be distinguished and identified based on the characteristic neutral loss of these groups
and their related fragments.
In mass spectrometry, peaks 19, 20, 31, 43, and 84 display identical ions at m/z 541
and 1083 in the primary mass spectrum. The m/z 541 ion disappears in the secondary mass
spectrum and is identified as [M−2H]2⁻, while m/z 1083 is identified as the [M−H]⁻ ion with
the molecular formula C48H28O30. The secondary mass spectrum reveals that these com-
pounds generate major fragment ions at m/z 601 [M−H−HHDP−glucose]⁻ and m/z 300 [el-
lagic acid−H]⁻, indicating significant neutral losses of HHDP and glucose. Peaks 19 and 20
also exhibit characteristic fragments at m/z 451 [Flavogallonic acid−H−H2O]⁻. Peak 19 is
identified as Punicacortein C and peak 20 as Punicacortein D based on the literature com-
parison. Peaks 31 and 43 are characterized by m/z 781 [M−H−HHDP]⁻ fragments. The re-
tention time and fragmentation pathway of peaks 31 and 43 were confirmed to correspond
to Punicalagin-α and Punicalagin-β; the fragmentation pathway of Punicalagin is showed
in Figure 8. Peak 84 is also characterized by m/z 449 [M−H−HHDP−glucose−galloyl]⁻, iden-
tified as T. chebula based on its retention time. The ions at m/z 542 and 1085 are observed
in the primary mass spectrum, while m/z 542 disappears in the secondary spectrum. These
are identified as [M−2H]2⁻ and m/z 1085 as [M−H]⁻ with the molecular formula C48H30O30.
Through the loss of HHDP and galloyl groups, the fragment ions m/z 783 [M−H−HHDP]⁻,
m/z 631 [M−H−HHDP−galloyl]⁻, and characteristic fragments at m/z 451 [Flavogallonic
acid−H−H2O]⁻ are generated. Based on ClogP values (Table S1) and the literature, peak 34
was identified as Rhoipteleanin G and peak 46 as Terflavin A. The ions at m/z 494 and 989
are observed in the primary mass spectrum, while m/z 494 disappears in the secondary
spectrum. These ions are identified as [M−2H]2⁻ and m/z 989 as [M−H]⁻, with the molecular
formula C41H34O29. The compound first loses a neoche group, forming the m/z 651
[M−H−neoche]⁻ fragment, and then loses a gallic acid to generate the m/z 481 [M−H−neo-
che−gallic acid]⁻ fragment. The characteristic fragment at m/z 337 [neochebulic
acid−H−H2O]⁻ is generated, and peak 58 is identified as Carpinusnin.
Molecules 2025, 30, 2451 23 of 32

Figure 8. The putative fragmentation pathway of Punicalagin.

Peaks 64, 67, 70, 74, 79, 87, 101, 109, 118, and 121 display ions at m/z 485 and 971 in
the primary mass spectrum. The m/z 485 ion disappears in the secondary mass spectrum,
while m/z 971 is identified as an [M−H]⁻ ion with the molecular formula C41H32O28. These
compounds are detected in the secondary mass spectrum through the loss of groups such
as gallic acid, HHDP, and neoche. The fragment ions observed include m/z 801 [M-H-
170]⁻, m/z 669 [M−H−302]⁻, m/z 633 [M−H−338]⁻, m/z 499 [M−H−170−302]⁻, m/z 463
[M−H−338–302]⁻, m/z 337 [neochebulic acid−H−H2O]⁻, and m/z 301 [ellagic acid−H]⁻. Based
on the chemical structure of components in Terminalia chebula, HHDP is likely attached to
the 3,6, 4,6, or 2,3 hydroxyl groups of glucose, while neoche groups generally do not attach
to the 1-hydroxyl group of glucose. Using ClogP values (Table S1) to assess polarity, the
following isomer structures were inferred and confirmed: Peak 64 corresponds to 4-gal-
loyl-6-neoche-2,3-HHDP-glucose and peak 67 to 1-galloyl-2-neoche-4,6-HHDP-glucose.
Peak 70 is identified as 1-galloyl-3-neoche-4,6-HHDP-glucose and peak 74 as 1-galloyl-2-
neoche-3,6-HHDP-glucose. Peak 79 corresponds to 1-galloyl-4-neoche-3,6-HHDP-glucose
and peak 87 to 1-galloyl-4-neoche-2,3-HHDP-glucose. Peak 101 is identified as 2-galloyl-
3-neoche-4,6-HHDP-glucose and peak 109 as 1-galloyl-6-neoche-2,3-HHDP-glucose. Peak
118 corresponds to 2-galloyl-4-neoche-3,6-HHDP-glucose and peak 121 to 6-galloyl-4-ne-
oche-2,3-HHDP-glucose.
In the primary mass spectrum, the ion signal at m/z 953 was observed in peaks 86, 90,
95, 105, 119, and 131, while the ion at m/z 476 was detected in peaks 86, 90, 95, 105, 119,
and 123, but not at peak 131. In the secondary mass spectrum, the signal at m/z 476 disap-
peared. Thus, m/z 476 was inferred to be the [M−2H]2⁻ ion peak, and m/z 953 was identified
as the [M−H]⁻ ion peak. The molecular formula was determined to be C41H30O27. Further
Molecules 2025, 30, 2451 24 of 32

analysis of the secondary mass spectrum indicates that the fragment patterns of peaks 86,
90, 95, 105, 119, and 123 are very similar. The main fragment ions observed include m/z
783 [M−H−170]⁻, m/z 651 [M−H−302]⁻, m/z 633 [M−H−320]⁻, m/z 481 [M−H−302−170]⁻, m/z
463 [M−H−320−170]⁻, m/z 337 [chebuloyl−H]⁻, m/z 331 [M−H−320−302]⁻, m/z 319
[M−H−302−170−162]⁻, and m/z 301 [ellagic acid−H]⁻. These fragment ions primarily result
from the loss of groups such as gallic acid, chebuloyl, HHDP, and glucose. Based on the
fragment ion information, six isomers were identified. Peak 105 was identified as chebu-
lagic acid by comparing its retention time and intensity with those of a reference standard;
the fragmentation pathway of chebulagic acid is showed in Figure 9. By assessing the po-
larity of each peak using ClogP values (Table S1), the following structures were assigned:
Peak 86 corresponds to 1-O-galloyl-3,4-chebuloyl-2,6-HHDP-D-glucose, and peak 90 cor-
responds to 1-O-galloyl-3,6-chebuloyl-2,4-HHDP-D-glucose. Peak 95 was assigned as 1-
O-galloyl-4,6-chebuloyl-3,3-HHDP-D-glucose and peak 119 as 1-O-galloyl-2,6-chebuloyl-
3,4-HHDP-D-glucose. Peak 123 was assigned as 1-O-galloyl-2,3-chebuloyl-4,6-HHDP-D-
glucose. The secondary fragments of peak 131 mainly include m/z 935.0799 [M−H−H2O]⁻,
m/z 917.0695 [M−H−2H2O]⁻, m/z 635.0896 [M−H−DHHDP]⁻, m/z 617.0781 [M−H−H2O−
DHHDP]⁻, m/z 465.0671 [M−H−DHHDP−gallic acid]⁻, and m/z 316.9932 [DHHDP−H]⁻. The
loss of DHHDP (318 Da) is the main neutral loss characteristic of the compound, suggest-
ing that peak 131 is terchebin.

Figure 9. The putative fragmentation pathway of chebulagic acid.

Peaks 113 and 115 display identical ions at m/z 492 and 985 in the primary mass spec-
trum. The m/z 492 ion disappears in the secondary mass spectrum, indicating that m/z 492
corresponds to the [M−2H]2⁻ ion, while m/z 985 is the [M−H]⁻ ion, with the molecular for-
mula determined as C42H34O28. In the secondary mass spectrum, fragment ions were ob-
served at m/z 815 [M−H−170]⁻, m/z 683 [M−H−302]⁻, m/z 633 [M−H−352]⁻, m/z 513
[M−H−302−170]⁻, m/z 463 [M−H−352−170]⁻, m/z 351 [6′-O-methyl neochebulic acid-H2O-
Molecules 2025, 30, 2451 25 of 32

H]⁻, and m/z 301 [ellagic acid−H]⁻. These ions are generated through the sequential loss of
gallic acid, HHDP, and 6′-O-methyl neochebuloyl. Based on the location of the galloyl
group, two isomers were deduced, and their polarity was determined using ClogP values
(Table S1). Peak 113 corresponds to 1-O-galloyl-3,6-HHDP-4-6′-methyl neochebuloyl-glu-
cose, while peak 115 corresponds to 2-O-galloyl-3,6-HHDP-4-6′-methyl neochebuloyl-glu-
cose.
Peaks 89, 93, 96, 103, 116, and 133 display identical ions at m/z 462 and 925 in the
primary mass spectrum. The ion at m/z 462 disappears in the secondary mass spectrum,
indicating that it corresponds to the [M−2H]2⁻ ion, while m/z 925 is assigned to the [M−H]⁻
ion. The molecular formula is determined to be C40H30O26. In the secondary mass spec-
trum, fragment ions were observed at m/z 773 [M−H−152]⁻, m/z 633 [M−H−292]⁻, m/z 481
[M−H−292−152]⁻, and m/z 465 [M−H−292−170]⁻, resulting from the sequential loss of gal-
loyl, THDP, and other groups. Based on the positions of the galloyl and THDP groups, six
isomers were deduced, and their polarity was determined using ClogP values (Table S1).
Peak 89 was identified as 1-O-galloyl-3,4-THDP-2,6-HHDP-D-glucose. Peak 93 was iden-
tified as 1-O-galloyl-2,4-THDP-6,6-HHDP-D-glucose (Phyllanthusiin C) and peak 96 as 1-
O-galloyl-3,6-THDP-2,4-HHDP-D-glucose. Peak 103 was identified as 1-O-galloyl-4,6-
HHDP-2,3-HHDP-D-glucose, while peak 116 was identified as 1-O-galloyl-2,3-HHDP-4,6-
HHDP-D-glucose. Peak 133 was identified as 1-O-galloyl-2,6-HHDP-3,4-HHDP-D-glu-
cose.

2.3.3. Terpenoids
Terpenoids generate abundant deprotonated molecular ions [M−H]⁻ in primary mass
spectrometry, with some also forming [2M−H]⁻ ions. These characteristics facilitate the
identification of excimer ions and the determination of their molecular formulas. The sap-
onins in Terminalia chebula are typically linked to glucose (Glc), galloyl, and glucoheptonic
acid through various substitution patterns. Aglycone fragments are relatively stable, pri-
marily losing sugar and galloyl fragments (152, 162, 208 Da) under normal voltages. By
analyzing the fragment ions of saponins, aglycones can be classified into 503 Da and 487
Da groups, each with multiple core configurations.
Peaks 139, 149, and 153 exhibit a high-intensity ion signal at m/z 711 in the primary
mass spectrum, identified as the [M−H]⁻ ion peak corresponding to the molecular formula
C37H60O13. In the secondary mass spectrum, the fragment at m/z 503 [M−H−208]⁻ displayed
a high-intensity signal, suggesting that it resulted from the loss of glucoheptonic acid.
Based on the saponin characteristics, the fragment at m/z 503 is attributed to the loss of
glucoheptonic acid. Peak 139 was identified as Arjungenin-24-O-glucoheptonic acid, and
peak 149 as Madecassic acid-24-O-glucoheptonic acid. Peak 153 was identified as Ter-
minolic acid-24-O-glucoheptonic acid. Peaks 141, 145, and 147 exhibit strong ion signals
at m/z 817 in the primary mass spectrum. Combined with secondary mass spectrometry,
m/z 817 is identified as the [M−H]⁻ ion peak, corresponding to the molecular formula
C43H62O15. The secondary mass spectrum reveals intense fragment ions at m/z 655
[M−H−162]⁻ and m/z 503 [M−H−162−152]⁻, likely resulting from the loss of glucose and
galloyl groups, as indicated by the characteristic fragments at m/z 503. Peak 141 was iden-
tified as Quercotriterpenoside I (Arjungenin-24-galloyl-28-glucose), peak 145 as
Madecassic acid-24-galloyl-28-glucose, and peak 147 as Terminolic acid-24-galloyl-28-glu-
cose. Peaks 158, 159, and 161 exhibit similar ion signals at m/z 503, 549, and 1007 in the
primary mass spectrum. The m/z 1007 peak is identified as the [2M−H]⁻ ion, while the m/z
549 peak corresponds to the [M+HCOO]⁻ ion, with a molecular formula of C30H48O6. Peak
158 was identified as Arjungenin, peak 159 as Madecassic acid, and peak 161 as Terminolic
acid.
Molecules 2025, 30, 2451 26 of 32

Peaks 148, 151, and 155 appear as [M−H]⁻ ion peaks at m/z 801, corresponding to the
molecular formula C43H62O14. The intense fragments at m/z 639 [M−H−162]⁻ and m/z 487
[M−H−162−152]⁻ in the secondary mass spectrum suggest that the characteristic fragments
at m/z 487 result from the loss of glucose and gallic acid groups. Peak 148 was identified
as Rotundic acid-24-galloyl-28-glucose and peak 151 as Asiatic acid-24-galloyl-28-glucose.
Peak 155 was identified as Arjunolic acid-24-galloyl-28-glucose. Peaks 150, 154, and 156
all appear as [M−H]⁻ ion peaks at m/z 695, with the molecular formula identified as
C37H60O12. In the secondary mass spectrum, an intense fragment at m/z 487 [M−H−208]⁻
was observed, which was attributed to the loss of glucoheptonic acid. Peak 150 was iden-
tified as Rotundic acid-24-O-glucoheptonic acid. Peak 154 was identified as Asiatic acid-
24-O-glucoheptonic acid and peak 156 as Arjunolic acid-24-O-glucoheptonic acid. Peak
160 exhibits a strong ion signal at m/z 639, corresponding to the molecular formula
C37H52O9. In the secondary mass spectrum, a fragment at m/z 487 [M−H−152]⁻ was de-
tected, attributed to the loss of a galloyl group. Peak 160 was identified as 23-galloyl-arju-
nolic acid. Peaks 162, 163, and 164 show ion peaks at m/z 487 and 975 in the primary mass
spectrum. The m/z 975 peak is identified as the [2M−H]⁻ ion, while m/z 487 corresponds to
the [M−H]⁻ ion, with a molecular formula of C30H48O5. Peak 162 was identified as Rotundic
acid. Peak 163 was identified as Asiatic acid and peak 164 as Arjunolic acid.

2.3.4. Other Components

Besides the previously mentioned ingredients, four additional compounds (2, 16, 47,
54, 69) have been identified in T. chebula. Details regarding their chemical compositions
are provided in the accompanying table.

2.4. The Applicability of the KID-NLF Strategy

The KID-NLF strategy has established identification methods for various medicinal
plants by summarizing the research of Moilanen et al. [60] and examining the mass spec-
trometry rules in T. chebula. Employing this strategy allows for the identification of gallic
acid derivatives, ellagitannins, and triterpenoids by precisely recognizing quasi-molecu-
lar ion peaks and their related secondary characteristic fragments. In addition to T. chebula,
plants like Phyllanthus emblica [61] and Punica granatum [62] (rich in tannins) as well as
Panax ginseng, P. quinquefolium, and P. notoginseng (containing triterpenoids) [63] also ad-
here to this identification rule. Further research has shown that iridoid glycosides, phe-
nolic acids, and flavonoids are compatible with this strategy. For instance, iridoid glyco-
sides dipsanosides A and B demonstrate specific mass spectrometry features, such as m/z
1519.519 [M+HCOO]−, 1473.514 [M−H]−, 759.252 [M+2HCOO]2−, and 736.249 [M−2H]2− as
adduct ions. Similarly, the phenolic acids isochlorogenic acid A, B, and C display peaks at
m/z 1029.229 [2M−H]−, 537.100 [M+Na−2H]−, and 515.118 [M−H]−, and the flavonoids hy-
peroside and isoquercitrin show ions at m/z 949.164 [2M+Na−2H]−, 927.182 [2M−H]−, and
463.089 [M−H]− [64,65]. Notably, alkaloids generate primarily [M+H]+ type quasi-molecu-
lar ions and lack distinctive adduct patterns, making them incompatible with the KID-
NLF strategy’s rule system for mass spectrometry. This finding indicates the need for
methodological validation based on component type when applying this strategy. Conse-
quently, it is advised to undertake methodological validation according to component
type when implementing this strategy and to formulate a specific identification strategy
for alkaloids.
Molecules 2025, 30, 2451 27 of 32

3. Experimental
3.1. Chemicals, Reagents, and Plant Materials
Deionized water was prepared using a Millipore Q purification system (Rephile,
Shanghai, China). HPLC-grade acetonitrile, methanol, and formic acid were obtained
from Sigma-Aldrich (Milwaukee, WI, USA). The crude medicinal materials derived from
dried, pitted mature fruits of T. chebula were procured from Chengdu in 2023. The author
identified these materials, and the specimens were deposited in the laboratory of the au-
thor. Reference products including shikimic acid (2), gallic acid (8), punicalagin α (31),
punicalagin β (43), corilagin (71), ellagic acid (88), chebulagic acid (105), chebulinic acid
(127), and 1,2,3,4,6-penta-O-galloyl-β-D-glucose (130) were purchased from Yuanye
(Shanghai, China), with purity exceeding 98% as determined by HPLC analysis.

3.2. Sample Preparation

Approximately 1 g of pitted and dried fruit powder of T. chebula powder, sieved
through a No. 3 mesh, was accurately weighed and transferred into a stoppered conical
flask. Subsequently, 50 mL of 70% (v/v) methanol was added with precision. The sealed
flask was weighed and then subjected to ultrasonic treatment at 250 W and 40 kHz for 30
min. After being allowed to cool, the flask was reweighed and any weight loss compen-
sated with 70% methanol. The supernatant was centrifuged at 13,000 rpm for 15 min and
filtered through a 0.22 µm microporous filter membrane.

3.3. UHPLC Analysis

UHPLC analysis was conducted using a Shimadzu LC-40 system (Shimadzu Corpo-
ration, Kyoto, Japan), which was equipped with an SPD-M40 detector. A Shim-pack GIST-
C18 column (2.1 × 100 mm, 2 µm) was utilized. The mobile phase consisted of 0.2% phos-
phoric acid aqueous solution (A) and acetonitrile (B). A gradient elution profile was em-
ployed as follows: 0–3 min, 3–6% B; 3–4 min, 6–6% B; 4–5 min, 6–7% B; 5–6 min, 7–7% B;
6–7 min, 7–15% B; 7–10 min, 15–15% B; 10–11 min, 15–21% B; 11–23 min, 21–21% B; 23–26
min, 21–33% B; 26–28 min, 33–35% B; 28–40 min, 35–35%. Detection wavelengths were
monitored at 254 nm. The flow rate was set at 0.4 mL/min, injection volume at 2 µL, and
column temperature at 30 °C.

3.4. UPLC-Q-TOF/MS Analysis

UPLC analysis was carried out using a 100 mm × 2.1 mm, 1.7 µm Waters Acquity
UPLCR BEH C18 column (Waters Corporation, Milford, MA, USA). The mobile phase
comprised acetonitrile (A) and water (B), each containing 0.1% formic acid. The linear
gradient program was set as follows: from 2% to 4% B over 0–2 min; from 4% to 5% B over
2–4 min; from 5% to 6% B over 4–11 min; from 6% to 9% B over 11–17 min; from 9% to
13% B over 17–21 min; maintained at 13% B over 21–27 min; from 13% to 60% B over 27–
32 min; from 60% to 99% B over 32–33 min; maintained at 99% B over 33–38 min; from
99% to 1% B over 38–40 min. The flow rate was set at 0.4 mL/min, injection volume at 1
µL, and column temperature at 40 °C.
The Waters SYNAPT G2HDMS system with an ion source was employed for elec-
trospray ionization (ESI). Scanning was conducted in negative (ESI−) ion mode, using ni-
trogen as the atomization and conical gas. The source temperature was set at 100 °C, and
the cone gas flow rate was maintained at 40 L/h. The desolvation temperature was held at
350 °C, and the gas flow rate was 800 L/h. Further MS settings included a sampling cone
voltage of 40 V, extraction cone voltage of 4 V, capillary voltage of 2.5 kV, scan time of 0.3
s, inter scan time of 0.02 s, and a mass-to-charge ratio (m/z) ranging from 100 to 1200.
Leucine-enkephalin (200 pg/mL) flowing at 10 µL/min was employed to calibrate the mass
Molecules 2025, 30, 2451 28 of 32

number m/z 554.2615. Data processing was conducted using MassLynx V4.2 and UNIFI
software (Version 1.9) from Waters Corporation, Milford, MA, USA.

4. Conclusions
In conclusion, a post-acquisition LC-MS data processing strategy, key ion diagnos-
tics–neutral loss filtering (KID-NLF), can effectively identify the structure of the natural
products responsible for the herbal extract. In this study, a total of 164 compounds were
identified by UPLC-Q-TOF/MS technique and KID-NLF strategy screening in 33 min run-
ning time, 47 of which were reported for the first time. This study provides a powerful
strategy for rapid profiling of chemical constituents of herbal medicines.

Supplementary Materials: https://www.mdpi.com/article/10.3390/molecules30112451/s1, Table S1:

The LogP and CLogP values of the chemical components in T. chebula were identified by UPLC-Q-
TOF/MS.

Author Contributions: J.Y.: writing—original draft preparation, drawing preparation, table ar-
rangement; X.Z.: drawing preparation, table arrangement; Y.H.: drawing preparation, table ar-
rangement; Y.Z.: funding acquisition, conceptualization, supervision. C.T.: conceptualization, writ-
ing—review and editing, supervision. All authors have read and agreed to the published version of
the manuscript.

Funding: The authors gratefully acknowledge the financial support from the National Key Research
and Development Program of China (No. 2023YFC3504400, 2023YFC3504401, 2023YFC3504402), the
Natural Science Foundation of Sichuan Province (2025ZNSFSC1821), and the “Xinglin Scholars”
Program of Chengdu University of TCM (CCYB2022009).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The datasets used and/or analyzed during the current study are avail-
able from the corresponding author on reasonable request.

Conflicts of Interest: The authors declare that they have no competing financial interests or personal
relationships that may have influenced the work reported in this study.

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