




































CTMJ | traditionalmedicinejournals.com 
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Abstract:  The goal of studying traditional Chinese medicine is to determine the biological roles played by the absorbed metabolites 

and prototypes in living organisms. However, due to endogenous interference and low metabolite abundance, complete in vivo 

component profiling is challenging. Thus, a methodical approach was suggested for the systematic screening and profiling of external 

components in biological matrices, using the mass differences between prototypes and their metabolic reaction products as a basis. 

Research Tools and Procedures: An extensive database based on metabolic reactions was constructed by collecting 247 metabolic 

reactions from the relevant literature on the Huangqi-Danshen (HD) herb pair. The mass differences were automatically computed 

using R programming and compared with the database in order to screen candidate components using the preprocessed data from 

experimental MS1 that was based on ultra-high-performance liquid chromatography combined with quadrupole-time-of-flight mass 

spectrometry. The components were then identified across the MS2 dataset. After oral administration of HD, 164 components were 

noted in the plasma samples of the rats. Out of them, 20 were validated using the reference standards. Amino acid dehydration, 

methylation, cysteine S-binding, glucuronidation, sulfation, and multistep reactions are prominent metabolic processes. Final 

Thoughts: This research paved the way for future studies to clarify HD's therapeutic benefits by revealing its metabolic features and 

biotransformation regulation. Beneficial tools for assessing metabolites in complex systems are provided by the suggested method. 

 

 

Chinese Traditional Medical Journal 
 

A Comprehensive Metabolite Profiling Approach Utilizing In vivo Metabolic Reaction-Based Mass 

Difference Screening with Ultra-High-Performance Liquid Chromatography and Quadrupole-Time-

of-Flight Mass Spectrometry for the Huangqi-Danshen Herb Pair 

S. Venkatesh, A. Meghana 
1,2 Academy of Acupuncture and Moxibustion, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 

350122, China 

Received on:  21 Jan 2025   Revised on: 20 Mar 2025   Accepted Date: 25 April 2025  

Published on: 18 May 2025 

 

 

 

 

 

INTRODUCTION 
 

Traditional Chinese medicines (TCMs) and their related 

biological roles may be better understood by examining how 

they are metabolized in complex systems. The absorbed 

prototype components may undergo metabolic processes to 

generate their equivalent metabolites in vivo after oral 

treatment [1]. Metabolic profiling of TCMs is still difficult, 

nonetheless, due to their  

complex molecular make-up and unique biological changes. 

the third The quantities of exogenous prototypes or their 

metabolites are often at trace levels and are readily disguised 

by background interference, in contrast to endogenous 

metabolites in biological matrices. [4] 

 

 

 

 

 



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Due to its broad application, high sensitivity, high 
mass accuracy, and plentiful fragment information, 
liquid chromatography coupled with high-resolution 
mass spectrometry has been extensively used for 
metabolic profiling of exogenous substances. Five, six 
Researches have devised typical methods for 
analyzing MS data, including diagnostic ion filtering, 
manual annotation with mass defect filtering, neutral 
loss filtering, and quick mining of exogenous 
components of biological matrices. Another technique 
to effectively annotate possible chemicals in 
complicated matrices is to build an internal database 
that matches the biotransformation rules of the 
chemicalome and metabolome using published 
metabolic pathways as templates. [10] Data analysis 
becomes tedious, time-consuming, and prone to 
errors due to the vast and repeated human 
procedures required by these methodologies. Optimal 
MS parameterization, preprocessing of raw data, and 
automated database creation have all been 
accomplished using programming tools like R, Python, 
and Java. on pages 11 and 12, An effective strategy 
for in vivo metabolic profiling is to construct feature 
extraction rules based on metabolic reactions during 
ultra-high-performance liquid chromatography 
combined with quadrupole-time-of-flight mass 
spectrometry (UHPLC-QTOF MS) analysis. References 
[13,14]:  
The constituents of the herb pair Huangqi-Danshen 
(HD), which includes Astragalus membranaceus 
(Fisch.) Bge. var. mongholicus (Bge.) Hsiao (Huangqi) 
and Salvia miltiorrhiza Bge. (Danshen), have been 
thoroughly studied in a preliminary study. These 
components primarily include phenolic acids, 
flavonoids, diterpenoids, and triterpenoids. However, 
the effects of these compounds in vivo remain 
unknown. By comparing the masses of metabolic 
reaction products and prototypes, we were able to 
devise a methodical approach to characterizing the 
exogenous components in rat plasma after oral HD 
treatment. We built a database after first collecting 
metabolic responses from the literature, then 
determining the mass changes between the prototype 
and the final result. Secondly, an ion list, containing 
RT and m/z values, was generated by preprocessing 
the full-scan MS1 data. After that, we used R to load 
the ion list and then used the mass differences to 
filter for potential components. The MS2 data on 
neutral losses, fragmentation modes, and diagnostic 
ions were used to further speculate or annotate the 
prototypes and their metabolites. We anticipated 
that this approach would provide HD in vivo 
metabolites, which would then serve as a reference 
for metabolic profiling of exogenous substances in 
TCMs and as a foundation for future studies on the 
pharmacodynamic basis and pharmacological 
processes. 

MATERIALS AND METHODS 
Chemicals and reagents 
Methanol and acetonitrile (HPLC‑grade) were purchased from 

Merck (Darmstadt, Germany). Analytical ethanol was purchased 

from Shanghai Titan Technology Co. Ltd. (Shanghai, China). 

Formic acid was supplied by ROE Company (Newark, USA). 

Isoflurane was supplied by Shenzhen Rivard Life Technology 

Co. Ltd. (Shenzhen, China). Heparin sodium was supplied 

by Shanghai Yuanye Biotechnology Co. Ltd. (Shanghai, 

China). Ultrapure water was prepared using a Milli‑Q water 

purification system (Millipore, Billerica, MA, USA). 

The A. membranaceus (Fisch.) Bge. var. mongholicus (Bge.) 

Hsiao was collected from Wulanchabu (Inner Mongolia, 

China), while S. miltiorrhiza Bge. was obtained from 

Linyi (Shandong, China). The herbal components were 

identified by Professor Hua Yang. A total of 76 chemical 

reference standards (purity > 95%) were used in this study, 

including 22 phenolic acids, 17 flavonoids, 19 diterpenoids, 

13 triterpenoid saponins, and 5 others. 

Standard solution and huangqi‑danshen samples 

preparation 
The A. membranaceus (Fisch.) Bge. var. mongholicus (Bge.) 

Hsiao and S. miltiorrhiza Bge. were mixed in a ratio of 

1:1 (w/w) and soaked in 50% ethanol (1:10, w/v) for 30 min, 

followed by extraction under reflux for 2 h twice. The extracted 

samples were mixed and concentrated under the vacuum 

using a rotary evaporator. Then, the concentrated extract was 

lyophilized and stored at 4°C. The lyophilized HD powder 

was dissolved in 1.5 g/mL saline before administration. The 

standard was dissolved in methanol (1.0 mg/mL) to obtain a 

single stock solution. 

Animal experiments 
All animal experiments were performed in compliance with the 

Guidelines for Pharmaceutical Animal Experiments of the China 

Pharmaceutical University (No. 2022‑03–047). Ten male Spraque-

Dawley rats (220 ± 20 g) were obtained from Vital River 

Laboratory Animal Technology Co., Ltd. (Zhejiang, China). 

The animals were kept in a constant chamber with a 12-h 

photoperiod, relative humidity (50 ± 5%), and temperature 

(25°C ± 2°C). They were allowed to eat and drink freely for 7 days 

to acclimatize. Before drug administration, all rats were allowed to 

drink freely for 12 h. 

The rats were randomly divided into HD and control groups. The 

HD group rats were administered by gavage at 6.25 g/kg, whereas 

the control group rats were administered normal saline. Blood 

samples were retrieved from ophthalmic veins using heparinized 

tubes at 0 (predose), 5, 15, 30 min, 1, 2, 

4, 6, 8, 10, 12, and 24 h. Immediately centrifuged the blood sample 

at 3000 rpm for 10 min at 4°C. The supernatant plasma at the same 

time point in rats was mixed in equal volumes and immediately 

stored at -80°C until analysis. 

Plasma sample and simulated biological sample preparation 



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After thawing on the ice, 90 L plasma samples were mixed 

with 360 L cold acetonitrile. The mixture was vortexed for 

3 min and centrifuged at 13,000 rpm for 15 min at 4°C. The 

supernatant was collected and dried using an EZ‑2 Personal 

Evaporator (GeneVac, UK). The metabolite extract was then 

dissolved in 90 L acetonitrile/water mixture (1:1, v/v), swirled 

for 3 min, and then centrifuged at 13,000 rpm at 4°C for 10 min 

before analysis. 

For the simulated biological sample, 120 L aliquots of blank 

plasma sample were placed to four 1.5 mL microcentrifuge 

tubes in equal parts after thawing on the ice. The obtained 

samples were mixed with cold acetonitrile (1:4, v/v), swirled 

for 3 min, and centrifuged at 13,000 rpm at 4°C for 15 min. 

The supernatant was collected in a microcentrifuge tube and 

dried using the EZ‑2 Personal Evaporator. To distinguish 

the isomeric reference materials, single reference material 

solutions were divided into four groups. The standard 

solution of each group was taken 5 L and prepared 

into 120 L mixed solution. The metabolite extract was 

reconstituted in the mixed solution and centrifuged at 

13,000 rpm for 15 min at 4°C after being vortexed for 3 min. 

The resulting supernatant was transferred to a sampling vial 

for UHPLC‑QTOF MS. 

Ultra‑high‑performance liquid chromatography coupled 
with quadrupole‑time‑of‑flight mass spectrometry 
analysis 
Detection was conducted using an Agilent 6530 

quadrupole time-of-flight mass spectrometer connected 

to an Agilent 1290 UHPLC (Agilent Technologies Inc., 

California, US). A Waters ACQUITY UPLC HSS T3 

column (2.1 mm × 100 mm, 1.8 m, US) was utilized at 

a column oven temperature of 35°C for chromatographic 

separation. The mobile phase made up of 0.1% (v/v) aqueous 

formic acid (phase A) and acetonitrile (phase B). The mobile 

phase gradient program was: 0–2 min, 5%–20% B; 2–7 min, 

20%–35% B; 7–9 min, 35%–50% B; 9–12 min, 50%–53% 

B; 12–18 min, 53%–60% B; 18–21 min, 60%–80% B; 

21–24 min, 80%–95% B; 24–30 min, 95% B while the 

posttime was 5 min. The volume injected was 2 L and flow 

rate was set to 0.3 mL/min. 

The mass spectrometric parameters were set as follows: drying 

gas, nitrogen; gas flow rate, 10 L/min; gas temperature, 350°C; 

nebulizer pressure, 35 psi; sheath temperature, 350°C; sheath 

gas flow, 11 L/min; electrospray capillary voltage, 4000 V in 

the positive mode and 3500 V in the negative mode; fragment 

voltage, 135 V; skimmer voltage, 65 V; octopole RF peak, 

750 V; mass range, MS: 100–1500 m/z, MS/MS: 50–1500 m/z; 

and collision energy, 10, 20, 30, 40, 50, and 60 eV. Mass 

spectrometry was conducted in the positive and negative ion 

modes. To prove the accuracy of mass spectrometry detection, 

the mass axis was calibrated by tuning the liquid before daily 

injection analysis. 

Database construction 
Through retrieving the relative literatures from PubMed (https:// 

pubmed.ncbi.nlm.nih.gov/) and Google scholar (https:// 

scholar.google.com/) using the keywords “Astragalus 

membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao, 

Huangqi, Salvia miltiorrhiza Bge., Danshen, Metabolites.” We 

collected the related metabolic reactions and drug components 

in vivo and organized the substituents created or lost during 

the metabolic reactions. Moreover, we manually recorded 

information on the mass differences created during the metabolic 

reactions. The database contains information on reaction names, 

formula changes, mass differences, classifications, and citations. 

Data processing 
Raw MS1 data for the blood samples were processed using 

Profinder B.10.0 (Agilent Technologies, USA). First, the data for 

HD group and control group were grouped according to the positive 

ion mode or negative ion mode with peak height ≥100 counts. 

The adduct ions of positive ions were [M + H]+, [M + Na]+, 

and negative ions were [M-H]−, [M + Cl]−, [M + HCOO]−. An 

isotope model was set for common organic compounds (no 

halogens). Other parameters were as follows: limit assigned 

charge states to a maximum of 1; RT window = 0.00% 

+0.4 min; mass window = 25 ppm + 2.0 mDa; compound ion 

count threshold ≥ 2. The EIC tolerance was set at ± 35.0 ppm 

and the RT was ± 1.5 min. The ion lists were then exported to 

Microsoft Excel files (. csv), which contained key information 

such as RT and mass. 

R‑assisted candidate components screening 
For the rapid screening of candidate components in the 

bio-samples, R programming (version 4.0.2) was used to 

develop the code. The ion lists were imported into the R 

programming to automatically calculate the mass differences 

between each pair of ions in the list to screen more candidate 

components. The mass differences between the experimental 

feature ions and the database were manually compared, and the 

candidate ions with mass error within ± 10 ppm were collected 

for further research. 

RESULTS 
The establishment of a systematic strategy 
A systematic strategy was developed to screen and analyze 

the exogenous components in biological matrices using 

the following procedure [Figure 1]. First, the raw MS1 

data were preprocessed using Profinder 10.0 software to 

generate ion lists, which included RT and mass values for 

detected components. Second, a mass‑difference database 

containing information on the biotransformation rules of 

the components was built to screen the metabolites in vivo. 

The mass differences between the experimental ions were 

calculated automatically using R programming, and the 

values were screened by matching them with the database. 

Subsequently, a mass error within 10 ppm was included 

for the candidate components. Finally, the prototype and 

metabolite constituents in the plasma were annotated and 



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Figure 1: The schematic diagram of the comprehensive strategy 

 

identified according to literature, reference standards, and 

MS2 information. 

Database for metabolic reaction of mass difference in 
huangqi‑danshen 
For drug compounds in vivo, a metabolic reaction of the mass 

difference database was established based on the prototypes 

and metabolites to screen feature ions quickly and effectively. 

By searching the reference literature from PubMed and Google 

Schola, 46 studies related to the identification of components 

of HD and related components in vivo were identified and 

organized. A total of 247 metabolic reactions were collected 

and the mass difference of each metabolic reaction was 

calculated manually. 

Database combined with R programming for screening 
of candidate components 
Profinder 10.0 was used to extract 862 positive and 976 negative 

ions from the original MS1 information. The feature ion lists were 

imported into the R programming to automatically calculate the 

mass difference between different ions. Then, the mass difference 

of each feature ion pair was matched with the database, and the ions 

pair of mass error within ± 10 ppm was collected as the candidate 

components. Consequently, 485 positive and 566 negative ions 

were efficiently screened as potential HD‑related candidates. 

Identification the absorbed prototypes and metabolites 
of huangqi‑danshen 
To identify the candidate HD components, MS2 data were 

collected for further analysis based on diagnostic ions, neutral 

losses, reference substances, and literature reports. The total 

ion chromatograms of rat plasma after oral HD treatment are 

shown in Figure 2. A total of 164 compounds, including 90 

prototypes and 74 metabolites, were explicitly or presumably 

identified in this study. The compounds included 49 flavonoids, 

28 phenolic acids, 84 terpenoids, and 3 others. Tables 1 and 2 

display the detailed information. 

Identification of flavonoids 
Flavonoids are the main components of Huangqi and can be 

further divided into flavones, isoflavones, flavanones, and 

flavonols.[16] They have many pharmacological activities 

such as promoting osteogenic function, improving liver cell 

dysfunction, and exerting anti‑inflammatory effects.[17-19] In 



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5 

2 2 

2 

 

 

Figure 2: Total ion chromatograms in positive (a) and negative (b) mode in rat plasma after oral administration of Huangqi‑Danshen 
 

this study, 49 flavonoids were annotated as HD, including 27 

prototypes and 22 metabolites. 
Identification of phenolic acids 
Phenolic acids, which mainly originate from Danshen, provide 

The component P22 (C 
16
H O ) generated an [M-H]− ion anti‑inflammatory, anti‑oxidant, anti‑thrombotic, and other 

pharmacological effects.[21-23] Salvianolic acid compounds 

at m/z 283.0604, which produced fragment ions at m/z 

239.0509 ([M-H-CO ]−), 237.0535 ([M-H-OCH -O]−), and 

225.0175 ([M-H-CO -CH ]−). Compared to the standard 

contain danshensu or caffeic acid in their basic structure, forming 

different dimers, trimers, tetramers, and salt derivatives.[24] In this 

2 2 study, 28 compounds were characterized in the plasma samples, 

reference, P22 was confirmed to be calycosin. In addition, P37 

showed an [M-H]− ion at m/z 267.0690 and an [M + H]+ ion at 

consisting of 14 prototypes and 14 products. 

m/z 269.0804, with the same formula as C H O . P37 exhibited Compound P8 generated a molecular formula of C
9
H

10
O

5 
and 

16  12  4 
− − exhibited [M-H]− ion at m/z 197.0472. The MS2 characteristic 

ions at m/z 252.0450 ([M-H-CH3] ), 223.0396 ([M-H-CO2] ), 

208.0578 ([M-H-CH -CO ]−), and 137.0418 (Retro Diels-Alder 

information at m/z 169.0313, 165.0652, 153.0233, and 151.0067 

3 2 correspond to [M-H-CO]−, [M-H-2O]−, [M-H-CO ]−, 

reaction, RDA reaction), which were determined to be 

formononetin using a standard reference. 

Metabolite M7 (C
22

H
23

NO
8
S) showed a parent ion at m/z 460.1088 

[M-H]−, and the product ions were generated at m/z 283.0624 and 

239.0174 matched those of P22. Compared to the literature, M7 

was identified as the metabolic product of P22, which is involved 

in di‑hydrogenation and cysteine 

2 

and [M-H-H O-CO]−, respectively. P8 was confirmed to 

be danshensu by comparison with a standard reference. Moreover, 

the prototype compounds P4 and P12 were characterized as 

protocatechuic and rosmarinic acids, respectively, based on 

comparison with the characteristic ions of the standards. 

Metabolite M1 (m/z 211.0618, [M + H]+) was 16 Da 

S-binding.[20] Metabolite M8 (C H
20 

O
10 

, m/z 443.1012, [M-H]−) larger than ferulic acid, with a molecular formula 

was eluted at 6.98 min and its product ions at m/z 

267.0761 ([M-H-C H O ]−), 252.0563 ([M-H-C H O -CH ]−) 

of C 
10

H
10

O
5
. The product ions were observed at m/z 

+ + 

6  8  6 6  8  6 3 
− 195.0588 ([M + H-O] ) and 167.0735([M + H-CO2] ), 

and 208.0438 ([M-H-C
6
H

8
O

6
-CH

3
-CO

2
] ), showing the 

same MS2 fragmentation pattern with P37. Therefore, M8 

suggested the presence of C
6
H

8
O

6
, which increased by 176 Da 

relative to formononetin [Figure 3]. Metabolite M30 (m/z 283.0958, 

[M + H]+) was 14 Da higher than that of P37, showing the same 

product ions at m/z 268.0954 and 239.0952 connected with [M + H-

CH ]+ and [M + H-CO ]+. Therefore, 

which had the same characteristics as ferulic acid. Therefore, 

M1 was hypothesized to be the hydroxylation product of ferulic 

acid.[25] Metabolite M9 displayed an [M-H]− ion at m/z 

273.0101, which was 62 Da higher than that of the parent 

danshensu, indicating the presence of sulfate group binding (+80 

Da), methylation (+14 Da), and dehydration (-18 Da) reactions. 

The MS2 fragment 
3 2 

M30 may be the methylation product of P37. Figure 4 shows the 

potential flavonoid metabolic pathways, including glucuronidation, 

methylation, cysteine S-conjugation, and hydrogenation. 

information for M9 at m/z 193.0821 and 229.0707 indicated the 

loss of SO3 (80 Da) and CO2 (44 Da), respectively. The 

fragmentation pathway is shown in Figure 5. In addition, this 

study mainly presents the metabolic reactions of related 

 
 

 

 
 

 

 
 

 

 

 

 a  
 
 
 
 
 
 
 
 
 
 
 

22 

12 



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22   

 

 
 
 

 
 
 
 
 
 
 
 
 

 

 
26 32  12 

 
 

 

17 14  6 
 

 

 
7  6  4 

 
 

 

10 10  4 
 
 

 

10 12  5 
 
 

 

16 14  4 

 
 

 

9  10  5 
 
 

 

21 20  11 
 
 
 
 
 
 

 
18 16  8 

 
 

 

22 22  9 
 
 
 
 
 

 

 
17 18  5 

 
 

 

23 28  11 
 
 
 
 
 

 

 
17 16  5 

 
 
 
 

 
23 24  11 

 

 
 
 

 
23 28  10 

 
 
 

 
16 12  5 

 
 
 

 

 
18 16  5 

 
 
 

 
41 68  14 

 
 
 
 

 
18 14  4 

 

 

 

18 16  5 
 
 

 
17 16  5 

 

Contd... 

Table 1: Identification of the prototype constituents of HD in plasma by UHPLC‑QTOF MS 

No. RT 
(min) 

m/z Adducts Formula Theoretical 
m/z 

Error 
(ppm) 

Fragment ions Identification Structure 
Type 

P1 2.23 341.0670 [M+H]+ C
18

H
12

O
7 341.0656 4.10 297.0788, 282.0495 Salvianolic acid G Phenolic acids 

P2 2.49 581.1850 [M+HCOO]− C H O 581.1876 -4.44 417.1187, 353.1311, 1-Hydroxypinoresinol-1-O- Phenolic acids 
       374.0591, β-D-glucoside  

P3 2.92 313.0693 [M−H]− C H O 313.0712 -6.07 285.0179, 251.0431, 4’,5-Dihydroxy-7,8- Flavonoids 

       137.0239 dimethoxyflavone  

P4* 3.17 153.0200 [M−H]− C H O 153.0193 4.36 135.0470, 123.0491, Protocatechuic acid Phenolic acids 

       109.0304   

P5 3.17 239.0567 [M+HCOO]− C H O 239.0561 2.51 215.0708, 203.0922, Caffeic acid methyl ester Phenolic acids 

       116.0545   

P6 3.55 211.0595 [M−H]− C H O 211.0612 -8.05 193.0578, 179.0236, Danshensu methyl ester Phenolic acids 

       169.0763, 150.0375   

P7* 3.65 269.0807 [M−H]− C H O 269.0819 -4.58 203.0848, 175.0246, Medicarpin Flavonoids 

       147.0134, 93.0354   

P8* 4.11 197.0480 [M−H]− C H O 197.0455 8.63 169.0313, 165.0652, Danshensu Phenolic acids 

       153.0233, 151.0067   

P9 5.60 447.0973 [M−H]− C H O 447.0933 8.38 295.0582, 131.008 Kaempferol 3‑O-β-D- Flavonoids 
        glucoside  

P10 5.62 461.1041 [M+H]+ C
22

H
20

O
11 461.1078 -8.11 285.0621, 71.0007 Wogonoside Flavonoids 

P11 5.63 461.1120 [M−H]− C
22

H
22

O
11 461.1089 6.65 177.0296, 85.0345 Hispiduloside Flavonoids 

P12* 6.44 359.0792 [M−H]− C H O 359.0767 5.46 302.1233, 283.0118, Rosmarinic acid Phenolic acids 
       235.0542   

P13* 6.81 431.1329 [M+H]+ C H O 431.1337 -1.76 369.0485, 271.0183, Ononin Flavonoids 

       269.0822, 241.0865,   

P14 7.31 489.1383 [M+H]+ C
24

H
24

O
11 

489.1391 -1.71 257.0051, 237.0712 Calycosin 7-O-β-D- 

glucoside-6’’-O-acetate 

Flavonoids 

P15* 7.35 303.1218 [M+H]+ C H O 303.1227 -2.97 285.1221, 199.0871, Isomucronulatol Flavonoids 
       125.0942, 107.0618   

P16 7.57 479.1585 [M−H]− C H O 479.1559 5.46 303.0291, 301.0472 (3R)-7,2’-Dihydroxy-3’,4’- Flavonoids 
        dimethoxyisoflavan‑7‑O-β-  

        D-glucuronide  

P17 7.64 501.1085 [M−H]− C
24

H
22

O
12 501.1038 9.28 235.0739, 193.0568 Di-feruloyl-tartaric acid Phenolic acids 

P18 7.69 301.1036 [M+H]+ C H O 301.1071 9.46 283.1046, 167.0715 (6aR,11aR)-3,9- Flavonoids 
        Dimethoxy-10-  

        hydroxypterocarpan  

P19 7.69 477.1378 [M+H]+ C H O 477.1391 -2.80 300.1300, 269.0956, Odoratin 7-O-β-D- Flavonoids 

       167.0688, 145.0899 glucopyranoside  

P20 7.74 373.0950 [M−H]− C
19

H
18

O
8 373.0929 5.65 249.0125, 135.06 Methyl rosmarinate Phenolic acids 

P21 7.77 463.1640 [M−H]− C H O 463.1610 6.69 164.0442, 153.0659 2’-Hydroxy-3’,4’- Flavonoids 
        dimethoxyisoflavan 7‑O-β-  

        D-glucoside  

P22* 8.01 283.0604 [M−H]− C H O 283.0612 -2.83 239.0509, 237.0535, Calycosin Flavonoids 

       225.0175, 183.0146   

P23* 8.17 283.0635 [M−H]− C
16

H
12

O
5 283.0612 8.13 269.0679, 196.0774 Glycitein Flavonoids 

P24* 8.44 313.1076 [M+H]+ C H O 313.1071 1.76 295.1090, 267.0986, Tanshindiol B Diterpenoids 
       257.0920, 253.0872,   

       219.0855   

P25* 8.84 829.4600 [M+HCOO]− C H O 829.4591 1.07 785.1802, 489.4880, Isoastragaloside IV Triterpenoids 

       315.6395   

P26 9.11 487.1263 [M−H]− C
24

H
24

O
11 487.1246 3.49 311.022, 145.0784 6’’-O-Acetylglycitin Flavonoids 

P27 9.32 295.0956 [M+H]+ C H O 295.0965 -3.00 277.0777, 267.0653, Trijuganone A Diterpenoids 

       259.0777, 251.0746   

P28 9.39 313.1061 [M+H]+ C H O 313.1071 -3.03 299.1282, 297.1072, Tanshindiol A Diterpenoids 
       267.1042, 205.0338   

P29 9.57 299.0919 [M−H]− C H O 299.0925 -2.00 269.0774, 255.0559 3-Hydroxy-4,9- Flavonoids 

        dimethoxypterocarpan  

 



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19 20  4 

 
 

 

18 22  3 
 
 

 

19 20  4 
 
 

 

19 18  4 
 
 
 
 
 
 

 
20 20  4 

 

 

 
10  13  5  5 

 

 

 

16 12  4 
 
 
 
 

 
17 16  3 

 
 

 

16 16  5 
 
 

 

17 18  3 
 
 
 
 
 
 

 
18 14  4 

 
 
 
 

 
18 20  5 

 

 

 
18 16  4 

 
 

 

 
18 18  5 

 
 

 
 
 
 

 
22  18  2  3 

 
 

 

19 20  3 
 
 

 

18 18  2 
 
 

 

17 18  4 
 
 

 

19 20  4 
 
 

 
19 18  4 

 

 
 
 

 
18 20  4 

 
 

 

20 20  5 
 
 

 
20 24  6 

 
 

 

18 22  3 

 
 

Contd... 

Table 1: Contd... 

No. RT 
(min) 

m/z Adducts Formula Theoretical 
m/z 

Error 
(ppm) 

Fragment ions Identification Structure 
Type 

P30 9.69 313.1431 [M+H]+ C H O 313.1434 -1.07 297.1280, 295.1333, 3-Hydroxycryptotanshinone Diterpenoids 

       285.1468, 271.1060   

P31 9.70 285.1515 [M−H]− C H O 285.1496 6.66 241.1436, 229.1076, Przewalskin F Diterpenoids 

       167.0387   

P32 9.91 313.1403 [M+H]+ C H O 313.1434 -9.90 298.1101, 269.1509, Miltionone I Diterpenoids 

       257.1604, 255.1087   

P33 9.92 311.1258 [M+H]+ C H O 311.1278 -6.38 297.1137, 267.1361, Hydroxytanshinone Diterpenoids 
       253.0781, 147.0768,   

       57.0704   

P34 9.99 293.1147 [M+H]+ C
19

H
16

O
3 293.1172 -8.60 275.1185, 219.0843 Dehydrotanshinone Diterpenoids 

P35 9.99 325.1415 [M+H]+ C H O 325.1434 -5.96 215.0817, 211.1118, (S)-5,7-Dihydroxy-6- Flavonoids 
       165.0656 prenylflavanone  

P36 10.00 284.0967 [M+H]+ C H N O 284.0989 -7.74 238.0934, 136.0534, Guanosine Others 

       135.1183, 107.0564   

P37* 10.39 267.0690 [M−H]− C H O 267.0663 9.18 252.0450, 223.0369, Formononetin Flavonoids 

       208.0578, 137.0418   

P38 10.46 293.0821 [M+H]+ C
18

H
12

O
4 293.0808 4.32 265.0849, 249.0946 Tanshinol A Diterpenoids 

P39 10.47 269.1183 [M+H]+ C H O 269.1172 4.01 251.1073, 233.0999, Epidanshenspiroketallactone Diterpenoids 
       223.1110, 205.1021   

P40 10.49 287.0956 [M−H]− C H O 287.0925 9.81 269.1250, 259.0922, (3R)-7,2’,3’-Trihydroxy-4’- Flavonoids 

       243.1326 methoxyisoflavane  

P41 10.70 271.1335 [M+H]+ C H O 271.1329 2.32 259.0857, 197.0723, Neocryptotanshione II Diterpenoids 

       179.0898   

P42 10.72 377.0792 [M+Cl]- C
19

H
18

O
6 377.0797 -1.33 311.0899, 297.0931, Tanshinoic acid B Phenolic acids 

       249.0577, 243.0766   

P43 10.76 295.0960 [M+H]+ C H O 295.0965 -1.69 277.0852, 251.0577, 3α‑Hydroxymethylenetansh Diterpenoids 

       237.0527, 209.0597 inquinone  

P44* 10.82 283.0605 [M−H]− C
16

H
12

O
5 283.0612 -2.47 253.0364, 237.0669 Wogonin Flavonoids 

P45 11.14 317.1403 [M+H]+ C H O 317.1384 6.15 301.1504, 299.1252, 7-O-methylisomucronulatol Flavonoids 
       285.1503, 271.1363   

P46 11.15 297.1131 [M+H]+ C H O 297.1121 3.25 279.1019, 265.1266, Tanshinol B Diterpenoids 
       261.0932, 233.0946,   

       179.0867   

P47 11.24 315.1217 [M+H]+ C H O 315.1227 -3.17 297.1102, 279.1140, 15,16-Dihydrotanshinol B Diterpenoids 

       271.1292, 147.0778   

P48 11.78 453.1319 [M+Cl]- C
22

H
26

O
8 453.1322 -0.66 399.1609, 181.0615, Syringaresinol Others 

       137.0081   

P49 11.79 359.1421 [M+H]+ C H N O 359.1390 8.63 342.1324, 341.1469, Isosalviamides G Diterpenoids 
       311.1269   

P50 11.80 297.1483 [M+H]+ C H O 297.1485 -0.74 271.1349, 249.1057, Isocryptotanshinone Diterpenoids 

       211.0768   

P51 11.82 267.1367 [M+H]+ C H O 267.1380 -4.87 253.1243, 249.1410, 4-Methylenemiltirone Diterpenoids 

       239.1465, 224.0543   

P52 11.82 287.1300 [M+H]+ C H O 287.1278 7.71 269.1116, 233.1072, 2’-Hydroxy-3’, Flavonoids 

       181.0981 4’‑dimethoxyisoflavan  

P53 11.83 313.1428 [M+H]+ C H O 313.1434 -1.92 123.1181, 267.1253, 17-Hydroxycryptotanshinone Diterpenoids 

       295.1341, 297.1212   

P54* 11.84 311.1286 [M+H]+ C H O 311.1278 2.62 295.0989, 293.1124, Tanshinone IIB Diterpenoids 

       267.1372   

P55 11.92 293.0797 [M+H]+ C
18

H
12

O
4 293.0808 -3.75 249.0883, 178.0775 Monodydroxytanshinone I Diterpenoids 

P56 11.92 301.1446 [M+H]+ C H O 301.1434 3.87 285.1441, 283.1330, Salvianonol Diterpenoids 

       265.1307, 255.0993   

P57 11.96 341.1362 [M+H]+ C H O 341.1389 -7.91 221.0972, 115.0953, Trijuganone C Diterpenoids 

       83.0277   

P58 11.97 359.1506 [M−H]− C H O 359.1500 1.67 221.0972, 115.0953, Lariciresinol Others 

       83.0277   

P59 12.03 287.1642 [M+H]+ C H O 287.1642 0.10 271.1628, 269.1544, Cryptoacetalide Diterpenoids 
       261.1322, 251.1406,   

       231.1016   

 



Table 1: Contd... 

CTMJ | traditionalmedicinejournals.com 
Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 

 

24   

 

 
 
 
 
 

 
20 26  2 

 
 

 
18 14  3 

 

 

 
16 12  6 

 
 
 

 

 
17 18  4 

 
 

 
19 18  4 

 
 

 

19 18  5 

 
 

 

18 16  4 
 
 
 

 
18 16  4 

 
 
 
 

 
25 24  12 

 

 
 
 

 
19 20  3 

 

 

 
18 20  2 

 
 

 
18 14  3 

 
 

 

18 14  3 
 
 

 

18 16  4 
 
 

 

29 26  12 
 
 

 

18 16  3 
 
 

 

19 22  4 
 
 

 
25 30  11 

 

 
 

 
18 22  2 

 

 

 
29 26  12 

 
 

 
 
 
 

 
19 20  3 

 
 

 

18 12  3 
 
 

 

19 18  3 
 
 

 

19 22  2 
 
 

 
20 24  2 

 

 

RT: retention time; *compound with a reference standard 

No. RT 
(min) 

m/z Adducts Formula Theoretical 
m/z 

Error 
(ppm) 

Fragment ions Identification Structure 
Type 

P60 12.43 299.1981 [M+H]+ C H O 299.2006 -8.21 284.1627, 255.1587, Microstegiol Diterpenoids 

       253.1972, 241.1245   

P61 12.49 279.0991 [M+H]+ C H O 279.1016 -8.85 267.1035, 265.0792, Methylenetanshinquinone Diterpenoids 

       233.0933, 221.0775   

P62 12.49 301.0707 [M+H]+ C H O 301.0712 -0.55 285.0849, 245.0820 3,3’,7-Trihydroxy-4’- 

methoxyflavone 

Flavonoids 

P63 12.49 579.1719 [M+H]+ C
27

H
30

O
14 579.1708 1.90 521.1344, 425.0752 Violanthin Flavonoids 

P64 12.93 309.1083 [M+Na]+ C H O 309.1097 -4.53 223.0723, 165.0588, 4’-Hydroxy-5,7- Flavonoids 

       139.0717 dimethoxyflavan  

P65 12.93 311.1249 [M+H]+ C H O 311.1278 -9.27 297.1037, 295.1289, Isotanshinone IIB Diterpenoids 

       283.0846, 237.1279   

P66 12.93 327.1199 [M+H]+ C H O 327.1227 -8.56 309.1136, 283.1298, 3-Hydroxytanshinone IIB Diterpenoids 

       265.1206, 223.0751   

P67* 12.97 297.1116 [M+H]+ C H O 297.1121 -1.8 281.1041, 279.1038, Danshenxinkun A Diterpenoids 
       263.0995, 251.1018,   

       233.1006   

P68 13.01 295.0994 [M−H]− C H O 295.0976 6.10 277.1000, 249.0971, Tanshinone VI Diterpenoids 

       237.0969   

P69 13.01 297.1123 [M+HCOO]− C
17

H
16

O
2 297.1132 -3.14 253.0741, 238.0611 Salyunnanin D Diterpenoids 

P70 13.67 517.1327 [M+H]+ C H O 517.1341 -2.71 471.1302, 455.1270, Formononetin-7-O-β-D- Flavonoids 
       343.1491, 103.0473 glucoside-6”-O-malonate  

P71 13.85 355.1032 [M+Na]+ C
14

H
20

O
9 355.1005 9.14 271.1145, 183.0568 Leonuriside A Phenolic acids 

P72 13.86 297.1474 [M+H]+ C H O 297.1485 -3.77 269.1572, 253.1582, 1-Oxomiltirone Diterpenoids 

       237.0902, 211.1415   

P73 13.86 269.1539 [M+H]+ C H O 269.1536 1.09 254.1205, 227.1181, Salvinone Diterpenoids 

       191.0002   

P74* 14.86 279.0996 [M+H]+ C H O 279.1016 -7.06 261.0978, 237.0809, Dihydrotanshinone I Diterpenoids 

       233.0947   

P75 15.21 279.0986 [M+H]+ C H O 279.1016 -9.46 261.0783, 233.0869, 1,2-Dihydrotanshinquinone Diterpenoids 

       221.0803   

P76 15.21 297.1138 [M+H]+ C H O 297.1121 5.60 279.1019, 261.0961, Salmiltiorin A Diterpenoids 

       233.1056   

P77 15.62 567.1486 [M+H]+ C H O 567.1497 -1.94 225.0348, 184.0741, Ethyl lithospermate Phenolic acids 

       135.0745   

P78* 15.70 281.1180 [M+H]+ C H O 281.1172 2.77 263.1048, 248.0706, Trijuganone B Diterpenoids 

       235.1119, 220.0855   

P79* 15.77 315.1561 [M+H]+ C H O 315.1591 -9.52 297.1489, 279.1381, Neocryptotanshinone Diterpenoids 

       251.1421   

P80 16.03 505.1690 [M−H]− C H O 505.1715 -4.95 243.0881, 241.1412, 2’-Hydroxy-3’,4’- Flavonoids 

       225.0823 dimethoxyisoflavan 7‑O-β- 

D-glucoside 6”-O-acetate 

 

P81 16.13 293.1496 [M+Na]+ C H O 293.1512 -5.46 265.1234, 261.1287, Miltiorolide A Diterpenoids 

       257.1442, 247.1139   

P82 16.35 565.1344 [M−H]− C H O 565.1351 -1.24 509.0867, 323.1432, Dimethyl lithospermate Phenolic acids 

       224.0818, 211.0551   

P83 16.52 521.1345 [M−H]− C
24

H
26

O
13 521.1301 8.44 313.0461, 141.0486 Salviaflaside Phenolic acids 

P84 18.22 313.1441 [M−H]− C
19

H
22

O
4 313.1445 -1.28 283.1308, 175.1814 Dehydrocrotonin Diterpenoids 

P85 18.41 297.1490 [M+H]+ C H O 297.1485 1.61 281.1305, 279.1459, Cryptotanshinone Diterpenoids 

       271.1259, 241.1491   

P86* 18.75 277.0860 [M+H]+ C H O 277.0865 -1.80 263.0643, 235.1056, Tanshinone I Diterpenoids 

       231.0628, 221.0914   

P87* 21.96 295.1305 [M+H]+ C H O 295.1329 -8.03 281.1139, 277.1146, Tanshinone IIA Diterpenoids 

       263.0981, 249.1241   

P88* 22.56 283.1670 [M+H]+ C H O 283.1693 -8.12 267.1591, 225.0886, Miltirone Diterpenoids 
       197.1239   

P89 22.68 319.1645 [M+Na]+ C H O 319.1669 -7.52 277.0877, 249.0788, Saprorthoquinone Diterpenoids 

       204.0850   

P90 24.70 367.1689 [M+Cl]- C
20

H
28

O
4 367.1682 1.91 334.1107, 271.0346 Pinophicin A Diterpenoids 

 



Table 1: Contd... 

CTMJ | traditionalmedicinejournals.com 
Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 

 

25   

 

 
 

 
 
 
 
 
 
 
 

 
 
 

 
11 11 9 

 
 

 
10 11 5 

 
 
 

 

 
18 10  10 

 
 
 

 

 
22 23 8 

 
 
 
 

 
10 10  7 

 

 

 

24 24  11 
 
 

 
23 26  12 

 
 
 

 

27 26  8 2 
 
 

 
24 22  10 

 

 

 
17 19 3 

 
 
 

 
18 22  4 

 
 

 

28 24  8 
 
 
 
 

 
 

 
20 26  5 

 
 

 

19 22  5 
 
 
 
 

 
19 25 12 

 

 

 
19 22  5 

 
 
 

 
 
 
 

 

 
19 23 5 

 
 

 

19 18  5 
 
 

 
17 14  4 

 
 
 
 

 
17 14  2 

 
 

 

17 18  4 

 

 

 

Contd... 

Table 2: Identification of the metabolite constituents of HD in plasma by UHPLC‑QTOF MS 

No. RT 
(min) 

m/z Adducts Formula Error 
(ppm) 

Fragment ions Identification Structure 
Type 

M1 1.35 211.0612 [M+H]+ C
10

H
10

O
5 5.21 195.0558, 167.0753, 105.0159 Ferulic acid-hydroxylation Phenolic acids 

M2 2.44 332.0076 [M−H]− C H NO S -1.73 287.0819, 285.0676, Unknown Phenolic acids 
      242.0637, 217.0203   

M3 2.49 226.0693 [M+H]+ C H O N -7.52 208.0462, 181.0514, Glycine conjugate of caffeic acid‑ Phenolic acids 

      149.0384, 137.0667, 135.0412 decarbonylation-hydroxylation  

M4 3.19 391.1017 [M−H]− C
19

H
20

O
9 -4.49 347.0351, 345.0284, 329.0401 Methyl-rosmarinic acid-hydration Phenolic acids 

M5 5.28 416.9955 [M−H]− C H O S 7.94 337.0324, 321.0465, Calycosin-dicarbonylation- Flavonoids 
      283.1038, 257.0786 dehydroxylation-sulfate  

M6 5.62 461.1041 [M+H]+ C
22

H
20

O
11 -8.11 446.1054, 285.0621 Calycosin-glucuronide Flavonoids 

M7 5.63 460.1088 [M−H]− C H NO S 3.56 416.1256, 283.0624, 239.0174 Cysteine S-conjugate of calycosin- Flavonoids 

       dihydrogenation  

M8 6.98 443.1012 [M−H]− C
22

H
20

O
10 6.39 267.0761, 252.0563, 208.0438 Formononetin-glucuronide Flavonoids 

M9 7.28 273.0101 [M−H]− C H O S 9.72 231.0957, 229.0707, Methyl-danshensu-dehydration-sulfate Phenolic acids 

      193.0959, 179.0928   

M10 7.32 487.1294 [M−H]− C H O 9.88 311.0935, 283.1167, 255.1111 (6aR,11aR)-3,9,10-Tri- Flavonoids 

       methoxypterocarpan-glucuronide  

M11 7.69 495.1550 [M+H]+ C H O 9.70 301.0928, 213.1757, 3-Hydroxy-9,10- Flavonoids 
      167.0701, 57.0861 dimethoxyptercarpan-glucuronide-  

       hydration  

M12 7.99 477.1562 [M−H]− C H O -2.29 415.0689, 321.0535, Dimethyl lithospermate-loss of 2CO Phenolic acids 
      245.0204, 224.0423   

M13 8.26 469.1185 [M−H]− C H O 9.55 293.0885, 277.0956, 3α‑Hydroxymethylenetanshinquinone‑ Diterpenoids 

      257.1204, 229.0877 glucuronide  

M14 9.01 286.1465 [M+H]+ C H NO 9.54 256.1095, 225.1297, 213.1322 Glycine conjugate of Diterpenoids 
       epidanshenspiroketallactone-  

       didecarbonylation-hydroxylation  

M15 9.01 303.1563 [M+H]+ C H O -9.19 301.1411, 287.0901, Salvianonol-reduction Diterpenoids 

      267.1429, 257.1430   

M16 9.10 487.1380 [M−H]− C H O -3.78 453.0858, 399.1635 Salvianolic acid-methyl ester- Phenolic acids 

       dehydroxylation  

M17 9.15 347.1953 [M−H]− C
16

H
30

NO
7 1.01 329.1461, 305.1073 Unknown Phenolic acids 

M18 9.15 347.1892 [M−H]− C
20

H
28

O
5 8.07 295.0911, 259.1822 Savialba acid-hydrogenation Phenolic acids 

M19 9.46 345.1734 [M−H]− C H O 7.68 301.1441, 159.1391 7-O-methylisomucronulatol- Flavonoids 
       dimethylation-hydrogenation  

M20 9.55 331.1515 [M+H]+ C H O -7.55 315.0708, 313.1389, Hydroxycryptotanshinone-hydration Diterpenoids 

      297.1187, 295.1233   

M21 9.56 457.1280 [M−H]− C
27

H
22

O
7 -2.79 281.0793, 265.0678, 233.0750 Unknown‑glucuronide Flavonoids 

M22 9.56 458.1296 [M−H]− C H NO -1.74 404.1332, 282.0887, Glycine conjugate of danshensu Phenolic acids 

      211.0635, 153.0436 methyl ester-methylation-glucuronide  

M23 9.71 331.1570 [M+H]+ C H O -9.06 313.1480, 297.1303, Hydroxycryptotanshinone-hydration Diterpenoids 

      295.1223, 277.1273   

M24 9.88 501.1550 [M−H]− C
29

H
26

O
8 -0.98 421.1530, 245.1015, 179.1661 Dimethyl lithospermate-dehydration Phenolic acids 

M25 9.91 353.1375 [M+H]+ C
21

H
20

O
5 -2.41 337.1291, 249.1284 Danshenol A-hydroxylation Diterpenoids 

M26 9.91 331.1552 [M+H]+ C
19

H
22

O
5 3.62 313.1695, 295.1259, 285.1579 Hydroxycryptotanshinone-hydration Diterpenoids 

M27 9.93 346.1632 [M+H]+ C H NO -4.91 311.1239, 283.1518, Glycine conjugate of 4’‑Hydroxy‑5,7‑ Flavonoids 

      217.0991, 215.0534 dimethoxyflavan‑hydrogenation  

M28 10.00 327.1206 [M+H]+ C H O -6.42 311.1177, 283.1185, 267.1339, Tanshinone IIB-hydroxylation Diterpenoids 

      255.1285   

M29 10.00 283.0957 [M+H]+ C H O 9.25 267.0869, 243.1028, Danshenxinkun A-demethylation Diterpenoids 

      241.0864, 239.1111   

M30 10.01 283.0958 [M+H]+ C
17

H
14

O
4 -2.42 268.0954, 239.0952, 223.0847 Formononetin-methylation Flavonoids 

M31 10.47 251.1091 [M+H]+ C H O 9.73 236.1147, 233.0841, 223.1078, Dihydrotanshinone I-decarbonylation Diterpenoids 

      219.1053, 205.0930   

M32 10.48 285.1125 [M−H]− C H O -2.57 243.0406, 173.0926, 135.0734 (3R)-7,2’,3’-Trihydroxy-4’- Flavonoids 
       methoxyisoflavane‑methylation‑  

       dehydroxylation  

 



CTMJ | traditionalmedicinejournals.com 
Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 

 

26   

 

 
 
 

 
 
 
 
 
 
 
 

 
17 20  5 

 
 

 
 
 
 

 
18 14  5 

 
 

 
19 22  5 

 
 

 

18 14  4 
 
 
 
 
 

 

 

14 14  4 
 
 
 

 
24 25 7 

 
 

 

21 19 2 
 
 

 

20 26  4 
 
 
 
 
 
 
 
 

 
24 24  9 

 
 

 

18 20  3 
 
 

 

19 18  4 
 
 

 

19 20  5 
 
 

 
19 18  4 

 
 
 
 

 
21 14  5 

 
 
 

 
 
 

 
24 24  9 

 
 

 

20 27 11 
 
 

 

21 30  10 
 
 
 

 
17 18  8 

 
 
 
 

 
19 20  5 

 
 

 

18 18  3 
 
 

 
18 18  4 

 
 

 
18 18 

 

Contd... 

Table 2: Contd... 

No. RT 
(min) 

m/z Adducts Formula Error 
(ppm) 

Fragment ions Identification Structure 
Type 

M33 10.48 303.1258 [M−H]− C H O 6.61 259.0820, 241.0250, 225.0305 (R)-3-(5-Hydroxy-2,3,4- Flavonoids 
       trimethoxyphenyl)-chroman-7-ol-  

       decarbonylation  

M34 10.49 477.2654 [M−H]− C
30

H
38

O
5 1.58 459.2495, 435.1606, 415.1041 Unknown Diterpenoids 

M35 10.57 309.0750 [M−H]− C H O -5.98 281.0871, 265.0920 3-Hydroxy-4,9-dimethoxypterocarpan- Flavonoids 

       dehydration-carbonylation  

M36 10.65 329.1388 [M−H]− C H O -1.97 287.1084, 285.1690, Przewalskin F‑carbonylation Diterpenoids 

      267.0917, 257.0927   

M37 10.76 295.0980 [M+H]+ C H O 5.13 279.1138, 277.0825,267.1074, Dihydrotanshinone I-hydroxylation Diterpenoids 
      265.0970   

M38 10.76 489.1742 [M−H]− C
25

H
30

O
10 -4.95 313.1620, 149.0526 Neocryptotanshinone‑glucuronide Diterpenoids 

M39 10.78 475.1952 [M+H]+ C
25

H
30

O
9 -2.23 299.1636, 281.1463, 193.1597 Normiltirone‑glucuronide Diterpenoids 

M40 10.82 247.0953 [M+H]+ C H O -4.80 233.0709,231.0961, 229.0855, (6aR,11aR)-3,9-Dimethoxy-10- Flavonoids 
      189.0628 hydroxypterocarpan- demethylation-  

       decarbonylation-hydroxylation  

M41 10.83 470.1298 [M−H]− C H NO S 4.05 416.0904, 311.1240 N‑acetylcysteine S conjugate of Diterpenoids 

       17-Hydroxycryptotanshinone  

M42 11.14 318.1481 [M+H]+ C H NO -2.37 304.1107, 300.0863, 278.1487 Salviadione-methylation- Diterpenoids 

       carbonylation-dehydroxylation  

M43 11.24 331.1883 [M+H]+ C H O -6.30 316.1387, 315.1490, Microstegiol-hydroxylation Diterpenoids 

      313.1725, 299.1958, 284.1378   

M44 11.25 459.1616 [M+H]+ C
24

H
26

O
9 -7.32 311.0986, 237.1277 3-Hydroxytanshinone IIB-glu Diterpenoids 

M45 11.66 455.1342 [M−H]− C
24

H
24

O
9 -1.22 279.1132, 263.1598, 199.0646 Danshenxinkun B-glucuronide Diterpenoids 

M46 11.67 455.1354 [M−H]− C
24

H
24

O
9 1.42 279.1048, 249.0947, 211.1134 Trijuganone B-glucuronide Diterpenoids 

M47 11.78 455.1349 [M−H]− C H O 0.32 441.1393, 425.1377 Calycosin 7-O-β-D-glucoside-6’’-O- Flavonoids 
       acetate-dehydroxylation  

M48 11.80 285.1513 [M+H]+ C H O 9.75 267.1340, 241.1364, Deoxyneocryptotanshinone- Diterpenoids 

      229.0932, demethylation  

M49 11.82 311.1285 [M+H]+ C H O 2.30 295.1448, 293.1273, Danshenxinkun A-methylation Diterpenoids 

      265.1226, 249.0917   

M50 11.82 329.1389 [M+H]+ C H O 1.67 311.1154, 269.1005, 267.1443, Tanshinone IIB-hydration Diterpenoids 
      239.1102   

M51 11.84 311.1294 [M+H]+ C H O 5.19 296.1383, 295.1222, Danshenxinkun A-methylation Diterpenoids 

      293.0733, 283.1355   

M52 11.93 329.1378 [M+H]+ C
19

H
20

O
5 -1.67 311.1168, 283.1365, 267.1186 Tanshinone IIB-hydration Diterpenoids 

M53 11.86 347.0910 [M+H]+ C H O -1.15 301.0718, 203.0743, 163.0736 Monodydroxytanshinone Diterpenoids 
       I-methylation-dicarbonylation-  

       dehydroxylation  

M54 11.97 357.1350 [M−H]− C
20

H
22

O
6 1.79 329.1602, 313.1465, 299.1015 Neocryptotanshinone‑carboxylation Diterpenoids 

M55 12.48 455.1375 [M−H]− C H O 6.03 279.1034, 265.1148, Trijuganone B-glucuronide Diterpenoids 

      261.0883, 251.1161   

M56 12.48 456.1505 [M−H]− C H NO -1.39 412.0751, 375.0837, 279.1235 N‑acetylcysteine S conjugate of Phenolic acids 

       methyl rosmarinate  

M57 12.60 441.1738 [M−H]− C H O -6.39 299.1807, 265.1354 (3R, 4R)-4, 7-hydroxy-2’, Flavonoids 
       3’‑dimethoxyisoflavane‑4’‑O-β-D-  

       glucoside-decarbonylation-hydration  

M58 12.93 351.1078 [M+H]+ C H O 1.01 335.1120, 307.0831, 297.068, 4’‑Hydroxy‑5,7‑dimethoxyflavan‑ Flavonoids 

      199.0493 hydroxylation  

M59 12.95 325.1166 [M−H]− C
12

H
22

O
10 7.93 311.1232, 282.1309, 265.0908 Unknown Diterpenoids 

M60 12.95 327.1227 [M−H]− C H O -3.35 285.1346, 241.1055 7-O-methylisomucronulatol- Flavonoids 
       carbonylation-dehydroxylation  

M61 12.95 281.1205 [M−H]− C H O 7.76 265.0982, 261.1283, Przewalskin F‑reduction Diterpenoids 

      251.0887, 239.1044   

M62 13.58 297.1123 [M−H]− C H O -3.14 253.0741, 179.0598 4’‑Hydroxy‑5,7‑dimethoxyflavan‑ Flavonoids 

       carbonylation-dehydroxylation  

M63 13.86 251.1427 [M+H]+ C H O -1.36 239.1393, 235.1523, Cryptotanshinone-dehydration- Diterpenoids 

      233.1370, 223.1133 decarbonylation  

 



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27   

 

4 

2 

2 

2 

19  22  5 

 
 
 

 
 
 
 
 
 
 
 

 
16 12  2 

 
 

 
20 18  10 

 
 

 

21 18  9 
 
 

 
22 20  11 

 
 

 

22 22  6 
 
 

 

26 20  7 
 
 

 

18 12  2 
 
 

 

20 20  4 
 
 

 

18 18  4 

 

 

RT: retention time 

18 28  6 

 

compounds, including dehydration, sulfation, methylation, 
and hydroxylation. 

Identification of terpenoids 
A total of 84 terpenoid-related compounds were detected on HD 

after oral administration, which included diterpenoids and 

triterpenoid saponins. Tanshinones are the main diterpenoids in 

vivo and exhibit various bioactivities, such as anti-cancer and 

anti‑pulmonary fibrosis activities.[26,27] 

According to a comprehensive analysis of the chemical 

composition of HD, tanshinones usually show a high response in the 

positive mode, and the most obvious feature of these compounds 

is the neutral loss of CO and H2O. Taking 

was speculated to be methyl-danshexinkun A [Figure 6]. M20 and 

M23 exhibited [M + H]+ ions at m/z 331.1515 and 331.1570, 

respectively, which confirmed the same molecular formula as C H 

O . MS2 information was obtained at m/z 313, 297, and 295, 

which indicated losses of 18 Da (-H2O), 28 Da (-CO), and 36 Da 

(-2H2O), respectively. The quasi-molecular ions were 18 Da larger 

than P30, suggesting that they may be the hydration products of 

P30. Hydroxylation, dehydroxylation, decarbonylation, 

methylation, hydration, and other reactions occur during terpenoid 

metabolism. 

 

DISCUSSION 

P30 (C 
19
H O ) as an example, the protonated [M + H]+ 

In this study, HD‑related metabolic profiling was used to 

ion was observed at m/z 313.1431. The characteristic information 

exhibited at m/z 297.1280 ([M + H-O]+), 295.1333 ([M + H-H 

O]+), 285.1468 ([M + H-CO]+), and 

271.1060 ([M + H-CO-CH ]+), which was considered to be 

hydroxycryptotanshinone.[28] P67 (m/z 297.1116, [M + H]+) 

systematically characterize the absorbed compounds and their 

products. In total, 90 prototypes and 74 biochemical products were 

identified in rats orally administered HD. The major components 

found in rats were flavonoids, phenolic acids, 

produced a molecular formula of C 
18
H 

16
O

4
, while the and terpenoids, indicating that these could be the potential 

characteristic fragment ions at m/z 281.1041 ([M + H-O]+), 

279.1038 ([M + H-H O]+), 263.0995 ([M + H-H O-O]+), and 

bioactive components of HD. 

2 2 Flavonoids are the main active components of Huangqi and 

251.1018 ([M + H-H O-CO]+) in MS2 information. Therefore, P67 

was identified as danshenxinkun A by comparison with the 

standard. 

are highly bioavailable and easily absorbed into the blood. 

Calycosin, the aglycone of calycosin-7-O-β-D-glucoside, which  

possessed  pharmacological  activities,  including 

Metabolite M49 (C 
19
H 

18
O

4
) presented a quasi-molecular anti-inflammatory  and  anti-hepatic  injury  effects.[29,30] 

ion at m/z 311.1285 ([M + H]+), corresponding to CH from 

P67. A range of unique fragment information were 

collected at m/z 295 .1448 ([M + H- O] +) , 

293.1273 ([M + H-H O]+), 265.1226 ([M + H-H O-CO]+), 

Calycosin-7-O-β-D-glucoside may lose a glucosyl group into 

calycosin and produced formononetin by dehydroxylation reaction. 

Calycosin can be metabolized by glucuronidation 

to produce calycosin-glucuronide metabolites with improved 
2 2 

and 249.0917 ([M + H-H O-CO ]+). Therefore, M49 bioavailability. In addition, two isoflavone glycosides (calycosin 
2 2 

2 

20 

Table 2: Contd... 

No. RT 
(min) 

m/z Adducts Formula Error 
(ppm) 

Fragment ions Identification Structure 
Type 

M64 13.86 237.0921 [M+H]+ C H O 4.61 223.1107, 209.0947, Danshenxinkun C-dehydroxylation Diterpenoids 

      205.0989, 194.0785   

M65 13.87 449.0588 [M−H]− C H O S 8.93 415.0845, 407.0618, Methyl rosmarinate-dehydration- Phenolic acids 

      405.0615, 359.0374 methylation-sulfate  

M66 13.87 413.0883 [M−H]− C H O 1.20 345.1053, 323.0604, Methyl rosmarinate-dicarbonylation- Phenolic acids 

      295.0321, 223.0262 dehydroxylation  

M67 13.87 459.0896 [M−H]− C H O -8.03 415.0881, 341.1108 Kaempferol 3‑O-β-D-glucoside- Flavonoids 
       carbonylation-dehydroxylation  

M68 14.15 381.1351 [M−H]− C H O 1.94 375.1008, 271.1079 Calycosin-7-O-β-D-glucoside- Flavonoids 

       dehydroxylation  

M69 15.79 443.1146 [M−H]− C H O 2.20 425.1021, 367.0838, Unknown Flavonoids 

      285.0845, 275.0290   

M70 20.29 261.0899 [M+H]+ C H O -4.24 246.0793, 233.1012, Tanshinone I-dehydroxylation Diterpenoids 
      219.0659, 205.0969   

M71 20.30 325.1461 [M+H]+ C H O 8.19 281.1305, 279.0786, Dehydromiltirone-carboxylation Diterpenoids 

      261.0901, 253.1611   

M72 20.64 299.1276 [M+H]+ C H O -0.62 284.0908, 281.1136, 15,16-Dihydrotanshinol B- Diterpenoids 

      191.1415, 141.0617 dehydroxylation  

M73 21.74 293.1421 [M+H]+ C
19

H
18

NO
2 3.65 277.0615, 269.0408, 235.0188 Salviadione-dehydrogenation Diterpenoids 

M74 22.24 341.1925 [M+H]+ C H O -9.86 307.1255, 299.1614, Militibetin A-hydration- Diterpenoids 
      241.1284, 165.1307 dihydroxylation  

 



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Figure 3: The MS/MS spectra of formononetin and its possible metabolite. (a) Formononetin; (b) Formononetin‑glucuronidation; (c) Possible fragment 
pathways 

 

7-O-β-D-glucoside-6’’-O-acetate and formononetin 7-O-β-D- 

glucoside-6”-O‑malonate) and a flavonoid glycoside (odoratin 7-O-β-

D-glucopyranoside) were detected, demonstrating that they could 

be directly absorbed in vivo. According to the metabolic 

information, glucuronidation and cysteine S‑conjugation are the 

major metabolic pathways. Some Phase I reactions, such as 

methylation, hydroxylation, dehydroxylation, and reduction, occurred 

during the metabolism of HD. 

The plasma that contained the medication had prototypical 

components and phenolic acid metabolites in it. With 

pharmacological characteristics including antithrombotic actions, 

the one-phenolic acid molecule known as danshensu is a powerful 

physiologically active chemical. [31] in Danshensu takes up 

residence in the  

enters the circulation and, by means of a dehydration process, 

generates caffeic acid; this acid may then be methylated to create 

caffeic acid methyl ester. It is via glycine conjugation processes, 

glucuronidation, sulfation, and methylation that Danshensu 

produces metabolites. Researching and characterizing the pertinent 

biotransformation products in rats that mostly experienced Phase II 

metabolic processes was based on these findings. 

Terpenoids, including diterpenoids and triterpenoids, are the most 

abundant compounds found in drug-containing plasma. A total of 

84 terpenoids were identified in vivo during HD, including 46 parent 

constituents and 38 metabolites. Tanshinones are the main 

diterpenoids found in Danshen. Cryptotanshinone is easily 

metabolized into tanshinone IIB, 

 

 
 

 

 
 

 

 
 

 

 

 

 a  
 
 
 
 
 
 
 
 
 
 
 

 
 b  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 c  



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Figure 4: Proposed metabolic pathways of some typical flavonoids in rat plasma after oral administration of Huangqi‑Danshen 

 

hydroxycryptotanshinone, and tanshinone I. Tanshinone IIB 

and hydroxycryptotanshinone can further form metabolites 

through hydration reactions. Tanshinone I is metabolized to 

danshexinkun A, which generates methylated metabolites. 

Saponins are generally recognized to have poor oral 

bioavailability. A related study of Huangqi in vitro shows that 

it contains a large amount of triterpene saponins,[32] but during 

in vivo metabolism, triterpene saponin compounds enter the 

blood to a lesser extent. In our study, isoastragaloside IV was 

the only triterpene saponin-related component detected in 

rat plasma. This may be due to the large relative molecular 

mass of the triterpenoid saponin constituents, which makes 

it more difficult for them to be absorbed in vivo, resulting in 

low bioavailability. 

As mentioned above, the absorbed components mainly 

consist of flavonoids, phenolic acids, and terpenoids, 

suggesting that these components are the major contributors 

to HD. A large number of multi-step metabolites were 

found in plasma, indicating that these HD compounds 

underwent abundant metabolic reactions in vivo. Therefore, 

conducting an in-depth and comprehensive metabolic 

process analysis of HD in combination with biological 

samples such as urine, tissue, and feces is necessary. 



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Figure 5: The MS/MS spectra of Danshensu and its possible metabolite. (a) Danshensu; (b) Methyl‑danshensu‑dehydration‑sulfate; (c) Possible fragment 
 

This study aimed to elucidate the pharmacodynamic 

underpinnings of HD and offer a scientific foundation 

for the sensible use of this drug in clinical research and 

associated new drug development. 

 

CONCLUSIONS 
In vivo, TCMs may be subjected to a wide variety of drug 

metabolic mechanisms. By shedding or binding groups, the 

prototypes absorbed in biological matrices are transformed 

into metabolites. Metabolites in close proximity to one 

another also undergo biotransformation. Manual processing 

of mass discrepancies is required by traditional metabolite 

identification methodologies.  

between distinctive ions, a process that is both tedious and 

time-consuming. One way to lessen the burden on data 

processors is to automate the calculation of the mass 

difference using R programming. Based on the mass 

differences between prototypes and their products using 

UHPLC-QTOF MS, this work developed a systematic 

technique to identify and profile the exogenous components 

in TCMs. Both potential prototype components and 

metabolites that underwent multistage reactions were 

effectively screened using this technique. This led to the 

discovery of 164 chemicals in rat plasma, including 90 

prototypes and 74 metabolites. Terpenoids, phenolic acids, 

and flavonoids are its primary constituents. In vivo 

component characterization of HD has established 

 a  
 
 
 
 
 
 
 
 
 
 

 
 b  

 
 
 
 
 
 
 
 
 
 
 
 
 

 
 c  



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 a  
 
 
 
 
 
 
 
 
 
 
 

 
 b  

 
 
 
 
 
 
 
 
 
 
 
 
 

 
 c  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Figure 6: The MS/MS spectra of Danshenxinkun A and its possible metabolite. (a) Danshenxinkun A; (b) Methyl‑danshenxinkun A; (c) Possible 
fragment pathways 

 

for elucidating its active ingredient groups against diseases. 

Simultaneously, this strategy may provide a feasible method 

for metabolic profiling of exogenous compounds in biological 

matrices. 
 

 



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