CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 17 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] CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 18 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 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 19 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 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 20 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 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 21 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 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 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 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 23 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 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 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 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 28 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 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 29 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. CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 30 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 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 3 31 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. 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