Identification and Analysis of Chemical Constituents and Rat Serum Metabolites in Gushuling Using UPLC-Q-TOF/MS Coupled with Novel Informatics UNIFI Platform.

Gushuling (GSL), a well-known hospital preparation composed of traditional Chinese medicine (TCM), has been widely used in the clinical treatment of osteoporosis (OP) for decades due to its remarkable therapeutic effect. However, the chemical constituents of GSL are still unclear so far, which limits the in-depth study of its pharmacodynamic material basis and further restricts its clinical application. In this study, we developed a strategy for qualitative analysis of the chemical constituents of GSL in vitro and in vivo. Based on the results of ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS) and the UNIFI informatics platform, the chemical constituents of GSL can be determined quickly and effectively. By comparing the retention time, accurate mass, and fragmentation spectrum of the compounds in GSL, a total of 93 compounds were identified or preliminarily identified, including flavonoids, terpenoids, phenylpropanoids, steroids, etc. Among them, nine compounds have been confirmed by standard substances, namely epimedin A, epimedin B, epimedin C, icariin, ecdysterone, calycosin, calycosin-7-glucoside, ononin, and ginsenoside Ro. Fragment patterns and characteristic ions of representative compounds with different chemical structure types were analyzed. At the same time, 20 prototype compounds and 42 metabolites were detected in rat serum. Oxidation, hydration, reduction, dehydration, glutathione S-conjugation, and acetylcysteine conjugation were the main transformation reactions of GSL in rat serum. In this research, the rapid method to characterize the in vitro and in vivo chemical constituents of GSL can not only be used for the standardization and quality control of GSL but also be helpful for further research on its pharmacodynamic material basis.

1. Introduction

Osteoporosis (OP) is a complex bone disease characterized by low bone density and impaired microstructure of bone tissue, leading to increased bone fragility and being prone to fractures [1]. At present, the treatment drugs for OP mainly include ingredients that inhibit bone resorption, such as bisphosphonates, calcitonin, and estrogen, and ingredients that promote bone formation, such as parathyroid hormone [2]. However, taking bisphosphonates for a long time will increase the incidence of complications such as osteonecrosis of the jaw [3,4]. Long-term use of estrogen will also increase the incidence of breast cancer, endometrial cancer, and other diseases [5,6]. Nowadays, the importance of traditional Chinese medicine (TCM) and Chinese herbal compound prescription that have a therapeutic effect on OP were gradually been recognized because of their safety and effectiveness [7-9].

Gushuling (GSL) is a TCM preparation in the hospital of the First Affiliated Hospital of Anhui University of Chinese Medicine, consisting of Herba Epimedii (Yinyanghuo), Radix Achyranthis Bidentatae (Niuxi), Radix Astragali (Huangqi), and Concha Ostreae (Muli) as recorded in the Chinese Pharmacopoeia (Table S1). GSL has a good effect on the management of various types of OP in clinical treatment, such as senile osteoporosis [10,11], postmenopausal osteoporosis [12], and diabetes secondary osteoporosis [13]. However, the chemical constituents of TCM and related preparations are complex and the active ingredients are not fully understood [14,15]. Although the chemical constituents of GSL have been previously reported, only two or three bioactive ingredients have been found in GSL [16,17]. These efforts failed to reflect the overall chemical constituents of GSL, which made it was difficult to comprehensively evaluate the quality of GSL. Therefore, there is an urgent need for a reliable and efficient analytical method to determine the chemical constituents of GSL and the effective ingredients that can enter the body to achieve the purpose of quality control. Fortunately, the combination of ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS) and the UINIF analysis platform can solve this problem to some extent.

In recent years, UPLC-Q-TOF-MS has been widely used in various fields such as qualitative analysis of TCM ingredients, serum pharmacochemistry, metabolomics, and new drug development due to its high resolution and high sensitivity [18-21]. As a powerful information processing platform, UNIFI can automatically extract the mass spectrometry data of the sample, determine the molecular formula, compare it with the compound in the database, which gives the possible cracking method of the compound according to the fragment information of the compound under high energy, and display the detailed information of the identified compound under the preset filter conditions [22,23]. The purpose of this study was to use the UPLC-Q-TOF-MS technology in conjunction with UNIFI analysis software to comprehensively analyze and identify the chemical constituents of hospital preparation GSL and to analyze the prototype compounds and metabolites in rat serum, which provides a basis for the in-depth research and quality control of its pharmacodynamic material basis.

2. Materials and Methods

2.1. Materials and Reagents

Herba Epimedii, Radix Achyranthis Bidentatae, Radix Astragali, and Concha Ostreae were purchased from Anhui Xiehecheng Co., Ltd. (Bozhou, China) and evaluated by Doctor Rongchun Han (College of Pharmacy, Anhui University of Chinese Medicine, Hefei, China). The standard substances of calycosin (Lot number: MUST-18120901), calycosin-7-glucoside (Lot number: MUST-19031920), ononin (Lot number: MUST-19041101), epimedin C (Lot number: MUST-19081310), icariin (Lot number: MUST-18091010), and ecdysterone (Lot number: MUST-18120209) were purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). Epimedin A (Lot number: JOT-10354), epimedin B (Lot number: JOT-10353), and ginsenoside Ro (Lot number: JOT-10448) were purchased from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, China). The purity of all standards was over 98.0%. Acetonitrile (HPLC grade; Lot number: SHBM1775) and methanol were purchased from Sigma Aldrich Trading Co., Ltd. (Shanghai, China). Formic acid (HPLC grade; Lot number: J2027129) was obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Ultrapure water was prepared by a Milli Q-Plus system (Millipore, Bedford, USA).

2.2. Standards and Sample Preparation

First, Herba Epimedii and Radix Achyranthis Bidentatae were mixed at the ratio of 5 : 4 and refluxed with 70% ethanol at 8-fold volume (11-fold volume for the first time) for 3 times for 2 h, respectively. We used a rotary evaporator to volatilize the ethanol in the extract to obtain a concentrated solution for use. Second, Radix Astragali and Concha Ostreae were mixed at the ratio of 5 : 2 and boiled with 9.5-fold and 8-fold volume ultrapure water for 1 h, respectively. Finally, the ethanol extract obtained in the first step was mixed with the water extract obtained in the second step and concentrated to obtain the GSL sample solution. Herba Epimedii refluxed with 70% ethanol at 8-fold volume (11-fold volume for the first time) for 3 times for 2 h, respectively. We used a rotary evaporator to volatilize the ethanol and water in the extract to obtain a sample solution of Herba Epimedii. The preparation method of Radix Achyranthis Bidentatae sample solution is the same as that of Herba Epimedii sample solution. Radix Astragali boiled with 9.5-fold and 8-fold volume ultrapure water for 1 h, respectively, and concentrated to obtain the Radix Astragali sample solution. An appropriate amount of sample solution was diluted with methanol, and the supernatant was taken and stored at 4°C. All the solutions were filtered through a 0.22 µm filter membrane before analysis.

Nine standard substances were completely dissolved in methanol. Before qualitative analysis, they were mixed to prepare a mixed standard solution with appropriate concentration and passed through a 0.22 µm filter membrane. All solutions were stored in a refrigerator at 4°C.

2.3. Animals Handling and Serum Samples Preparation

Sprague Dawley rats (200 ± 20 g) of specific pathogen-free grade were purchased from Animal Experiment Center, Bengbu Medical College. Approved by the Experimental Animal Management and Ethics Committee of Bengbu Medical College, animal experimental research meets relevant ethical requirements. They were randomly divided into blank groups and GSL extract groups, with 6 rats in each group. The rats were kept in an animal room with a suitable environment for 7 days before the experiment. Rats in the blank group and GSL extract group were intragastrically administered distilled water and 51.993 g·kg−1·d−1 GSL sample solution for consecutive 3 days, respectively. The preparation process of the GSL sample solution is the same as that in the Standards and Sample Preparation section, and there is no need for subsequent operations such as methanol treatment. Before the last oral administration, the rats fasted for 12 h with free drinking water. Blood samples (500 µL) were collected from the fundus venous plexus 15 min after oral administration on the 3rd day and centrifuged for 10 min at 3500 rpm·min−1 at 4°C, and the supernatant was taken. Then, 1200 µL methanol was added to the 300 µL serum samples, vortexed, and centrifuged at 13000 rpm for 10 min. The supernatant was put into another tube and dried with nitrogen gas. The remaining was stored in acetonitrile (200 µL) and frozen at −80°C until analysis.

2.4. Chromatography and Mass Spectrometry Conditions

Chromatographic analysis was performed using a Waters Acquity™ UPLC system (Waters Corporation, Milford, USA). Chromatographic separation was carried on Waters ACQUITY UPLC® BEH C18 (2.1 × 100 mm, 1.7 µm) column by gradient elution with the optimal mobile phase of 0.1% formic acid aqueous solution (solvent A) and acetonitrile (solvent B), the column temperature was maintained at 35°C, and the temperature of the sample chamber was set to 8°C. The gradient elution was set as follows: 0−5 min, 5%–19% B; 5–10 min, 19% B; 10–11 min, 19%–25% B; 11–16 min, 25% B; 16–17 min, 25%–31% B; 17–22 min, 31%–51% B; 22–50 min, 51%–100% B; 50–55 min, 100% B; 55–60 min, 100%–5% B; 60–65 min, 5% B. The flow rate was 0.15 mL·min−1, and the injection volume was 2 µL.

A Waters Xevo G2 Q-TOF mass spectrometer (Waters Corporation, Milford, USA) equipped with an electrospray ionization (ESI) source operating in both positive and negative ion modes was connected to the UPLC. The full scan data were collected from m/z 50 to m/z 1200. For positive and negative ion modes, the capillary and cone voltage were set to 3.0 kV, 40 V and 2.5 kV, 40 V, respectively. The temperature of the conservation gas was set to 350°C, and the flow rate was set to 600 L·h−1. The two ion source temperatures in positive ion and negative ion modes were set to 120°C and 110°C, respectively. The cone gas flow rate was set to 50 L· h−1, and leucine and enkephalin were used as calibration fluids to ensure accuracy and repeatability.

2.5. UNIFI Data Processing Method

The chemical constituents analysis strategy was mainly divided into the following three steps:

The establishment of the chemical constituents library of GSL: The complete information on the compounds of three herbal medicines (Herba Epimedii, Radix Achyranthis Bidentatae, and Radix Astragali) in GSL was collected and obtained by searching China National Knowledge Infrastructure (CNKI), PubMed, PubChem, Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), ChemSpider, and other databases. The self-built compound library was established, including compound name, molecular formula, chemical structure (saved in “mol” format), accurate molecular mass, and other information. This information was imported into UNIFI. Among them, a total of 354 compounds were listed. When analyzing rat serum samples, the self-built database imported into UNIFI was the GSL in vitro compound information database that has been analyzed and confirmed.

Preliminary analysis of the results: We imported the original files of GSL sample solution, blank sample solution, rat administration serum, and blank serum analyzed by UPLC-Q-TOF-MS into the UNIFI software for samples comparison. Based on the automatic matching function of UNIFI software, compounds can be quickly identified. The parameter settings were as follows: analysis time range, 1–65 min; quality allowable error range, ±10 ppm; quality testing range, 50 Da to 1500 Da; positive adducts including H+, Na+, and K+; and negative adducts containing H−, HCOO−, and Cl−.

Manual review: By the MassLynx workstation, the above identification results were reviewed in combination with the precise mass of excimer ions, retention time, fragment ion information, and literature.

3. Results and Discussion

3.1. Chemical Constituents Analysis of Three Herbal Medicines in GSL

The separate extraction method of the three herbal medicines in GSL (Herba Epimedii, Radix Achyranthis Bidentatae, and Radix Astragali) was the same as the sample preparation process. Then, the TIC of the three herbal medicines in positive and negative ion modes were qualitatively analyzed according to the above-mentioned method, and the results are shown in Figure S1.

3.2. Identification and Analysis of Chemical Constituents in GSL

High-resolution mass spectrometry data of GSL were rapidly acquired by the UPLC-Q-TOF-MS method. The TIC of GSL in positive and negative ion modes is portrayed in Figure 1. The UNIFI screening platform was used to process and analyze its mass spectrometry data, and then the data were spontaneously matched with the fragment information. After further manual verification, it was found that there were 93 in vitro chemical constituents in the GSL recipe, including 51 kinds of flavonoids, 18 kinds of terpenoids, 6 kinds of phenylpropanoids, 5 kinds of steroids, and 13 others. Among them, 55 compounds were from Herba Epimedii, 23 compounds from Radix Achyranthis Bidentatae, and 21 compounds from Radix Astragali. In addition, Herba Epimedii and Radix Achyranthis Bidentatae contain four common compounds, and Herba Epimedii, Radix Achyranthis Bidentatae, and Radix Astragali contain one identical compound (rutin). Of the identified components, 9 compounds were identified by comparison with the standard substance. The detailed information on these components is gathered in Table 1 and Figure 2.

Figure 1

The total ion chromatography (TIC) of GSL from UPLC-Q-TOF-MS analysis. (a) Positive ion mode. (b) Negative ion mode.

Table 1

Identification of chemical constituents of GSL by UPLC-Q-TOF-MS.

NO.	Observed RT (min)	Formula	Observed m/z	Mass error (ppm)	Adducts	Fragment (+/−)	Identification	Structure class	Origin
1	1.63	C6H12O5	209.0670	1.3	[M+HCOO]−	89.0255	RAM	Saccharide	HQ
2	1.64	C5H10O5	195.0510	−0.2	[M+HCOO]−	89.0255	XLS	Saccharide	HQ
3	1.66	C23H24O11	475.1287	8.6	[M-H]−	89.0255, 179.0563, 290.0864, 341.1084	7,2′-Dihydroxy-3′,4′-dimethoxyisoflavone-7-O-β-D-glucoside	Flavonoids	HQ
4	1.67	C6H12O6	203.0522	−2.2	[M+Na]+, [M+K]+	127.0388	Inositol	Others	YYH
5	1.68	C10H14O3	205.0836	0.4	[M+Na]+	127.0388	3,4,5-Trimethoxytoluene	Others	YYH
6	1.69	C27H32O12	593.1879	0.6	[M+HCOO]−	89.0255, 179.0563, 290.0864, 341.1084, 377.0850	Maohuoside A	Flavonoids	YYH
7	1.76	C21H22O7	431.1381	7.8	[M+HCOO]−	128.0362	Wanepimedoside_qt	Flavonoids	YYH
8	5.25	C16H18O9	353.0867	−3.1	[M−H]−	191.0556	Chlorogenic acid	Phenylpropanoids (phenylpropionic acid)	YYH
		C16H18O9	355.1041	4.9	[M+H]+, [M+Na]+	163.0387	Chlorogenic acid	Phenylpropanoids (phenylpropionic acid)	YYH
9	6.44	C19H30O8	431.189	−6.6	[M+HCOO]−	163.0409, 173.0463, 219.0629	Icariside B1	Terpenoids (monocyclic monoterpenoids)	YYH
10	6.49	C19H28O10	461.1704	8.6	[M+HCOO]−	219.0629	Icariside D1	Terpenoids (monocyclic monoterpenoids)	YYH
11	6.87	C26H36O12	539.2149	2.7	[M−H]−	317.0238, 479.0789, 491.1857	Icariside E1	Phenylpropanoids (phenylpropanols)	YYH
12	6.92	C21H20O13	479.0807	−5.1	[M−H]−	316.0208	Isomyricitrin	Flavonoids	YYH
13	7.22	C32H38O15	697.1844	−8.8	[M+Cl]−	191.0563, 219.0647, 593.1515	Ikarisoside B	Flavonoids	YYH
14	7.86	C22H22O10	491.1183	−2.5	[M+HCOO]−	283.0601	Calycosin-7-glucoside∗	Flavonoids	HQ
		C22H22O10	447.1279	−1.4	[M+H]+	285.0749	Calycosin-7-glucoside∗	Flavonoids	HQ
15	7.92	C21H20O12	463.0849	−7.1	[M−H]−	285.0396, 431.0962	Hyperoside	Flavonoids	YYH NX
16	8.00	C15H10O7	303.0520	6.8	[M+H]+	287.0543	Robinetin	Flavonoids	YYH
17	8.17	C28H34O12	597.1798	9.1	[M+Cl]−	193.0869, 219.0659, 237.0750, 399.1276	Caohuoside D	Flavonoids	YYH
18	8.39	C27H44O7	525.3065	−0.8	[M+HCOO]−, [M+Cl]−	159.1057, 193.0869, 219.0659	Ecdysterone∗	Steroids	NX
		C27H44O7	481.3134	−5.5	[M+K]+, [M+Na]+	371.2244, 445.2923	Ecdysterone∗	Steroids	NX
19	8.42	C22H32O11	517.1889	−7.2	[M+HCOO]−, [M−H]−, [M+Cl]−	177.0907, 471.1833	Eugenol rutinoside	Phenylpropanoids (Styrene)	YYH
20	10.13	C29H38O15	625.2131	−1.1	[M−H]−	261.0762, 279.0868, 291.0852	Isomucronulatol-7,2′-di-O-glucosiole	Flavonoids	HQ
21	11.14	C16H12O5	283.0599	−4.7	[M−H]−	268.0381	Wogonin	Flavonoids	NX
		C16H12O5	285.0769	4.0	[M+H]+	270.0515	Wogonin	Flavonoids	NX
22	12.07	C32H38O16	677.2074	−1.9	[M−H]−	353.0993, 514.1457, 515.1522, 557.1635	Hexandraside E	Flavonoids	YYH
		C32H38O16	679.2231	−0.3	[M+H]+, [M+Na]+	299.0524, 355.1186, 517.1787	Hexandraside E	Flavonoids	YYH
23	12.18	C43H68O14	853.4619	3.3	[M+HCOO]−, [M−H]−	96.9603, 451.3268	1-O-{(3β,5ξ,9ξ)-3-[(6-Methyl-β-D-glucopyranuronosyl)oxy]-28-oxoolean-12-en-28-yl}-β-D-glucopyranose	Triterpenoids	NX
24	12.41	C29H50O	453.3471	−4.8	[M+K]+	343.3055	Sitosterol	Steroids	YYH NX
25	12.81	C38H48O20	825.2805	−0.8	[M+H]+	299.0541, 355.1165, 517.1669, 663.2131	Diphylloside A	Flavonoids	YYH
		C38H48O20	869.2693	−3.2	[M+HCOO]−, [M−H]−, [M+Cl]−	146.9657, 353.1024, 514.1468, 661.2136, 807.2701	Diphylloside A	Flavonoids	YYH
26	13.06	C37H46O19	793.2564	0.4	[M−H]−, [M+Cl]−, [M+HCOO]−	285.0389, 431.0974, 631.2028	Epimedoside D	Flavonoids	YYH
		C37H46O19	795.2681	−3.2	[M+H]+	287.0541, 299.0538, 355.1168, 517.1700	Epimedoside D	Flavonoids	YYH
27	13.29	C37H44O17	805.2492	−8.5	[M+HCOO]−	146.9657, 353.1024, 514.1468, 661.2136	Epimedoside	Flavonoids	YYH
28	13.32	C38H48O19	807.2703	−1.8	[M−H]−	146.9657, 353.1024, 514.1468, 661.2136	Diphylloside B	Flavonoids	YYH
		C38H48O19	831.2681	−0.2	[M+Na]+	121.0269, 299.0541, 355.1166, 547.1760	Diphylloside B	Flavonoids	YYH
29	13.39	C32H38O15	707.2195	0.3	[M+HCOO]−, [M−H]−, [M+Cl]−	146.9657, 353.1024, 514.1468	Epimedoside A	Flavonoids	YYH
		C32H38O15	663.2275	−1.2	[M+H]+, [M+Na]+	121.0269, 299.0541, 355.1166, 547.1760	Epimedoside A	Flavonoids	YYH
30	13.41	C26H30O11	517.1680	−6.9	[M−H]−	219.0671, 327.1270, 383.1110	(2S,3S)-3,5-dihydroxy-2-(4-hydroxyphenyl)-8-(3-methyl-2-buten-1-yl)-4-oxo-3,4-dihydro-2H-chromen-7-yl β-D-glucopyranoside	Flavonoids	YYH
31	14.13	C22H22O9	475.1212	−7.0	[M+HCOO]−	267.0642, 353.0994	Ononin∗	Flavonoids	HQ
		C22H22O9	431.1325	−2.8	[M+H]+	269.0797	Ononin∗	Flavonoids	HQ
32	15.81	C12H22O6	301.1069	6.9	[M+K]+	167.0713	3-Hexenyl-β-glucopyranoside	Saccharide	YYH
33	16.05	C16H12O5	283.0604	−2.9	[M−H]−	148.0159, 268.0364	Calycosin∗	Flavonoids	HQ
		C16H12O5	285.0751	−2.3	[M+K]+, [M+Na]+	270.0508	Calycosin∗	Flavonoids	HQ
34	17.76	C39H50O19	883.2882	0.6	[M+HCOO]−, [M−H]−, [M+Cl]−	146.9654, 251.0307, 529.1697, 675.2292	Epimedin A∗	Flavonoids	YYH
		C39H50O19	839.2961	−0.9	[M+K]+, [M+Na]+	313.0700, 369.1326, 531.1852	Epimedin A∗	Flavonoids	YYH
35	18.46	C38H48O18	853.2774	0.2	[M+HCOO]−, [M−H]−, [M+Cl]−	146.9656, 366.1101, 551.1510, 645.2186	Epimedin B∗	Flavonoids	YYH
		C38H48O18	809.2855	−0.9	[M+H]+, [M+K]+	313.0698, 369.1322, 531.1849, 677.2419	Epimedin B∗	Flavonoids	YYH
36	18.88	C21H20O6	369.1322	−2.9	[M+H]+	243.0640, 313.0699	Icaritin	Flavonoids	YYH
37	18.93	C39H50O19	867.2924	−0.5	[M+HCOO]−, [M−H]−, [M+Cl]−	146.9660, 366.1099, 551.1497, 659.2339	Epimedin C∗	Flavonoids	YYH
		C39H50O19	823.3014	−0.6	[M+H]+, [M+K]+	129.0545, 313.0699, 369.1322, 515.1931, 531.1852	Epimedin C∗	Flavonoids	YYH
38	18.99	C22H24O7	439.1143	−2.5	[M+K]+, [M+Na]+	239.0905, 301.0677	3,5,7-Trihydroxy-8-(3-methoxy-3-methylbutyl)-2-(4-methoxyphenyl)-4H-chromen-4-one	Flavonoids	YYH
39	19.17	C41H52O21	881.3114	4.6	[M+H]+	85.0294, 135.0437, 243.0645, 313.0698, 369.1321	Hexandraside B	Flavonoids	YYH
		C41H52O21	879.2921	−0.8	[M−H]−	267.0300, 367.0621, 451.0672, 513.1100, 613.1210	Hexandraside B	Flavonoids	YYH
40	19.21	C27H30O11	575.1754	−2.9	[M+HCOO]−, [M−H]−	175.0025, 351.0861, 367.1169, 513.1748	Icariside I	Flavonoids	YYH
		C27H30O11	531.1850	−2.1	[M+H]+, [M+Na]+	85.0294, 135.0437, 243.0645, 313.0698, 369.1321	Icariside I	Flavonoids	YYH
41	19.22	C33H40O15	721.2337	−1.7	[M+HCOO]−, [M+Cl]−	175.0025, 281.0437, 367.1169, 451.0672, 513.1748	Icariin∗	Flavonoids	YYH
		C33H40O15	677.2433	−1.1	[M+H]+, [M+K]+	135.0437, 313.0698, 369.1321, 531.1851	Icariin∗	Flavonoids	YYH
42	19.23	C20H24O6	399.1215	2.7	[M+K]+	135.0437	Lariciresinol	Phenylpropanoids (lignans)	HQ
43	19.26	C28H24O13	569.1236	−9.4	[M+H]+	85.0294, 135.0437, 313.0698, 369.1321	8-Hydroxy-6-methyl-9,10-dioxo-9,10-dihydro-1-anthracenyl 6-O-(3,4,5-trihydroxybenzoyl)-β-D-glucopyranoside	Anthraquinones	NX
44	19.63	C25H30O8	481.1785	−9.9	[M+Na]+	387.1427	Rubschisantherin	Phenylpropanoids (lignans)	NX
45	20.82	C36H42O17	747.2483	−1.5	[M+H]+	299.0532, 355.1114	Korepimedoside A	Flavonoids	YYH
		C36H42O17	745.2332	−2.3	[M−H]−	121.0304, 352.0934, 367.1168, 499.1620, 583.1812	Korepimedoside A	Flavonoids	YYH
46	20.94	C41H52O21	879.2892	−4.1	[M−H]−	367.1156, 381.0964, 571.1715, 673.2076	Korepimedoside C	Flavonoids	YYH
47	21.01	C56H88O25	1199.5264	1.5	[M+K]+	369.1320, 383.1110, 882.4549, 1059.5290	Achyranthoside D trimethyl ester	Triterpenoids	NX
48	21.08	C52H84O19	1013.5723	4.3	[M+H]+	295.0576, 311.0891, 385.1281, 882.4549	Chikusetsusaponin V butyl ester	Triterpenoids	NX
49	21.09	C53H82O25	1117.5026	−4.1	[M−H]−	312.0607, 383.1097, 529.1652, 631.2021, 997.4956	Achyranthoside D	Triterpenoids	NX
50	21.11	C47H74O18	949.4763	−0.5	[M+Na]+	295.0576, 311.0891, 882.4549	Chikusetsusaponin IV	Triterpenoids	NX
		C47H74O18	925.4757	−4.9	[M−H]−	139.1120, 171.1024, 211.1335, 229.1434, 367.1184	Chikusetsusaponin IV	Triterpenoids	NX
51	21.19	C48H76O19	955.4884	−2.5	[M−H]−, [M + HCOO]+	352.0934, 367.1168, 631.2021, 793.4236	Ginsenoside Ro∗	Triterpenoids	NX
52	21.24	C31H36O14	631.2021	−1.8	[M−H]−	121.0304, 352.0934, 367.1168, 583.1812	Ikarisoside F	Flavonoids	YYH
		C31H36O14	633.2146	−5.0	[M+H]+	299.0526, 369.1327, 385.1281	Ikarisoside F	Flavonoids	YYH
53	21.28	C45H60O24	1023.3186	7.8	[M+K]+	299.0526, 369.1327, 531.1847, 915.3315	Acuminatoside	Flavonoids	YYH
54	21.31	C27H30O11	529.1669	−8.8	[M−H]−	121.0304, 219.0656, 383.1102, 513.1729	Wushanicariin	Flavonoids	YYH
55	21.56	C35H42O16	763.2415	−5.2	[M + HCOO]−	367.1184, 381.0975, 555.1833, 645.2117	Sagittatoside C	Flavonoids	YYH
		C35H42O16	719.2591	6.3	[M+H]+	313.0706, 369.1324, 383.1134, 385.1288, 531.1848	Sagittatoside C	Flavonoids	YYH
56	21.61	C46H74O14	873.4927	−5.0	[M+Na]+	729.3908	Taibaienoside IV	Triterpenoids	NX
57	21.78	C20H18O6	353.1015	−4.3	[M−H]−	252.0419	8-Prenylkaempferol	Flavonoids	YYH
		C20H18O6	355.1173	−0.9	[M+H]+	253.0480, 269.0803, 299.0550	8-Prenylkaempferol	Flavonoids	YYH
58	21.79	C16H12O4	267.0656	−2.4	[M−H]−	252.0419	Formononetin	Flavonoids	HQ
		C16H12O4	269.0803	−2.0	[M+H]+, [M+Na]+	118.0413, 253.0480	Formononetin	Flavonoids	HQ
59	22.23	C17H16O5	301.1080	3.3	[M+H]+	167.0694	(6aR,11aR)-9,10-dimethoxy-6a,11a-dihydro-6H-benzofurano[3,2-c]chromen-3-ol	Flavonoids	HQ
60	22.33	C47H72O20	955.4528	−1.7	[M−H]−	382.1079, 645.2166, 835.4451, 925.4768	Achyranthoside C	Triterpenoids	NX
		C47H72O20	995.4167	−8.2	[M+K]+	272.2571, 299.0537, 383.1132	Achyranthoside C	Triterpenoids	NX
61	23.03	C32H38O14	645.2190	0.2	[M−H]−, [M+Cl]− [M+HCOO]−	223.0282, 366.1100	Sagittatoside B	Flavonoids	YYH
		C32H38O14	647.2329	−0.7	[M+H]+, [M+Na]+	191.0005, 207.0315, 313.0700, 369.1321	Sagittatoside B	Flavonoids	YYH
62	23.54	C17H20O6	343.1163	3.2	[M+Na]+	131.0491	1,2-Bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol	Phenylpropanoids (phenylpropionic acid)	YYH
63	23.89	C27H30O10	513.1762	−0.7	[M−H]−	146.9659, 217.0478, 351.0867, 366.1100	Icariside II	Flavonoids	YYH
		C27H30O10	515.1900	−2.2	[M+H]+, [M+K]+, [M+Na]+	135.0434, 243.0638, 313.0698, 369.1322	Icariside II	Flavonoids	YYH
64	23.90	C21H20O6	369.1321	−3.2	[M+H]+	135.0434, 243.0638, 313.0698	Anhydroicaritin	Flavonoids	YYH
65	24.53	C42H66O14	793.4358	−2.7	[M−H]−, [M+HCOO]−	209.0450, 367.1171	Zingibroside R1	Triterpenoids	NX
66	24.58	C19H30O9	437.1603	4.3	[M+Cl]−	209.0450	Icariside B3	Terpenoids (Monocyclic monoterpenoids)	YYH
67	25.14	C41H64O12	793.4391	1.5	[M+HCOO]−	469.3326, 487.3421	Acetylastragaloside I_qt	Triterpenoids	HQ
68	25.55	C20H18O5	337.1068	−4.0	[M−H]−	282.0493	Yinyanghuo D	Flavonoids	YYH
		C20H18O5	339.1227	0.0	[M+H]+	283.0583	Yinyanghuo D	Flavonoids	YYH
69	25.84	C25H26O6	421.1666	2.2	[M−H]−	351.1189	Yinyanghuo B	Flavonoids	YYH
		C25H26O6	423.1829	6.5	[M+H]+	349.1055	Yinyanghuo B	Flavonoids	YYH
70	26.15	C18H32O3	297.2423	−0.3	[M+H]+	119.0855	13-Hydroxy-9,11-octadecadienoic acid	Organic acids	HQ
71	26.23	C41H62O15	793.4010	−0.8	[M−H]−	631.3795, 673.3972	Achyranthoside C_qt	Triterpenoids	NX
72	26.71	C45H56O23	1003.2895	5.1	[M+K]+	313.0709	Korepimedoside B	Flavonoids	YYH
73	26.94	C27H44O7	481.3162	0.4	[M+H]+	251.1605	Inokosterone	Steroids	NX
74	28.62	C16H30O2	277.2149	4.1	[M+Na]+	223.1695	2,15-Hexadecanedione	Others	YYH
75	29.06	C25H24O6	421.1631	−3.6	[M+H]+	367.1135	Yinyanghuo A	Flavonoids	YYH
76	29.63	C21H20O5	351.1228	−2.9	[M−H]−	293.0440	Artonin U	Flavonoids	YYH
		C21H20O5	353.1355	−8.2	[M+H]+	297.0770	Artonin U	Flavonoids	YYH
77	29.76	C37H60O10	699.3821	−8.5	[M+Cl]−	277.2148, 353.2030	AstragalosideII_qt	Triterpenoids	HQ
78	30.20	C25H26O5	405.1700	−1.8	[M−H]−	295.0594	8,3′-diprenylapigenin	Flavonoids	YYH
		C25H26O5	407.1848	−1.2	[M+H]+	149.0233, 295.0598, 351.1224	8,3′-diprenylapigenin	Flavonoids	YYH
79	32.96	C16H22O4	301.1402	−2.7	[M+Na]+, [M+H]+, [M+K]+	149.0231	DBP	Others	NX
80	37.98	C30H48O3	455.3506	−5.3	[M−H]−, [M+HCOO]−	277.2164	Oleanolic acid	Triterpenoids	YYH NX
81	38.19	C18H30O2	279.2309	−3.4	[M+H]+	95.0854	Linolenic acid	Organic acids	HQ
82	38.31	C22H22O11	507.1170	5.2	[M+HCOO]−, [M+Cl]−	223.0281	Rhamnocitrin-3-O-glucoside	Flavonoids	HQ
		C22H22O11	501.0842	9.7	[M+K]+	191.0004, 207.0315, 281.0502	Rhamnocitrin-3-O-glucoside	Flavonoids	HQ
83	41.16	C18H32O2	281.2470	−1.9	[M+H]+	95.0852	EIC	Organic acids	HQ
84	44.22	C21H18O11	447.0959	8.4	[M+H]+	191.0001	Baicalin	Flavonoids	NX
85	46.85	C47H78O18	931.5353	9.8	[M+H]+	115.0034, 147.0651, 553.3685	Asernestioside A	Triterpenoids	HQ
86	46.86	C27H30O16	655.1574	8.8	[M+HCOO]−	91.0223, 223.0270	Rutin	Flavonoids	YYH NX HQ
		C27H30O16	633.1487	9.6	[M+Na]+, [M+H]+, [M+K]+	115.0034, 147.0651, 190.9999, 207.0309, 355.068	Rutin	Flavonoids	YYH NX HQ
87	48.41	C49H80O19	1011.4944	1.8	[M+K]+	147.0656, 184.0726, 621.3171	Asernestioside B	Triterpenoids	HQ
88	49.83	C17H14O6	315.0886	7.2	[M+H]+	207.0301, 281.0486	Jaranol	Flavonoids	HQ
89	50.62	C35H60O6	621.4377	0.8	[M+HCOO]−, [M+Cl]−	277.2198	Daucosterol	Steroids	YYH NX
90	52.31	C33H42O15	717.2087	−9.5	[M+K]+	221.0835, 429.0876	Wanepimedoside A	Flavonoids	YYH
91	57.66	C20H36O2	309.2763	−8.2	[M+H]+	89.0612	Linoelaidyl acetate	Others	YYH
92	57.75	C29H38O16	665.2115	9.5	[M+Na]+	133.0854	5′-Hydroxyiso-muronulatol-2′,5′-di-O-glucoside	Saccharide	HQ
93	57.90	C29H48O	413.3796	4.3	[M+H]+	95.0869	Stigmasterol	Steroids	NX

YYH: Herba Epimedii; NX: Radix Achyranthis Bidentatae; HQ: Radix Astragali; Compared with a reference standard.

Figure 2

Chemical structures of compounds identified in GSL.

3.3. Identification and Analysis of Chemical Constituents in Rat Serum

When analyzing the rat serum samples of GSL, the selected database was a self-built database constructed from analyzed and confirmed GSL in vitro compound information. The analysis steps of GSL in vivo components were the same as in vitro processing. The results showed that there were 20 chemical constituents in rat serum, including10 kinds of flavonoids, 4 kinds of triterpenoids, 2 kinds of phenylpropanoids, 1 kind of steroid, 1 kind of organic acid, and 2 others. Among them, 12 compounds were from Herba Epimedii, 8 compounds from Radix Achyranthis Bidentatae, and 2 compounds from Radix Astragali. At the same time, rutin is a common compound of three Chinese herbal medicines. Five compounds were identified by the standard substance. The specific information is shown in Table 2 and Figure S2.

Table 2

The chemical constituents of the GSL blood prototype identified by UPLC-Q-TOF-MS.

NO.	Observed RT (min)	Formula	Observed m/z	Mass error (ppm)	Adducts	Fragment (+/-)	Identification	Structure class	Origin
1	5.26	C16H18O9	353.0846	−9.2	[M−H]−	93.0350	Chlorogenic acid	Phenylpropanoids (phenylpropionic acid)	YYH
2	8.40	C27H44O7	481.3201	8.5	[M+H]+	175.1189, 288.2025	Ecdysterone∗	Steroids	NX
3	12.30	C43H68O14	853.4568	−2.8	[M+HCOO]−	137.0231, 312.1296	1-O-{(3β,5ξ,9ξ)-3-[(6-Methyl-β-D-glucopyranuronosyl)oxy]-28-oxoolean-12-en-28-yl}-β-D-glucopyranose	Triterpenoids	NX
4	12.72	C38H48O20	823.2644	−2.7	[M−H]−	214.9992, 312.1294	Diphylloside A	Flavonoids	YYH
5	13.01	C37H46O19	793.2537	−3.0	[M−H]−	631.2019	Epimedoside D	Flavonoids	YYH
6	13.34	C32H38O15	661.2083	−8.3	[M−H]−, [M+HCOO]−	353.0997	Epimedoside A	Flavonoids	YYH
7	17.58	C39H50O19	883.2807	−8.0	[M+HCOO]−	675.2247	Epimedin A∗	Flavonoids	YYH
8	18.44	C38H48O18	853.2745	−3.2	[M+HCOO]−, [M+Cl]−	252.0406, 366.1081, 645.2177	Epimedin B∗	Flavonoids	YYH
		C38H48O18	809.2861	−0.2	[M+H]+	369.1333, 531.1842	Epimedin B∗	Flavonoids	YYH
9	18.92	C39H50O19	867.2915	−1.6	[M+HCOO]−	366.1059, 659.2331	Epimedin C∗	Flavonoids	YYH
		C39H50O19	823.3031	1.5	[M+H]+	136.0765, 531.1840	Epimedin C∗	Flavonoids	YYH
10	19.20	C33H40O15	721.2341	−1.2	[M+HCOO]−, [M+Cl]−	367.1163	Icariin∗	Flavonoids	YYH
		C33H40O15	677.2413	−4.0	[M+H]+	313.0683, 369.1314, 531.1844	Icariin∗	Flavonoids	YYH
11	19.45	C25H30O8	481.1812	−4.3	[M+Na]+	97.0644	Rubschisantherin	Phenylpropanoids (lignans)	NX
12	21.12	C53H82O25	1117.5037	−3.2	[M−H]−	217.0817, 365.2313, 413.1985, 585.2846, 785.4175	Achyranthoside D	Triterpenoids	NX
13	21.21	C48H76O19	955.4937	3.0	[M−H]−	413.1985, 585.2846, 785.4175	Ginsenoside Ro∗	Triterpenoids	NX
14	21.92	C47H72O20	955.4622	8.2	[M−H]−	353.2213, 405.2627	Achyranthoside C	Triterpenoids	NX
15	22.99	C32H38O14	645.2157	−4.9	[M−H]−	223.0270	Sagittatoside B	Flavonoids	YYH
16	23.89	C27H30O10	513.1739	−5.3	[M−H]−	366.1082	Icariside II	Flavonoids	YYH
		C27H30O10	515.1908	−0.8	[M+H]+	313.0671	Icariside II	Flavonoids	YYH
17	28.61	C16H30O2	277.2140	0.6	[M+Na]+	223.1695	2,15-Hexadecanedione	Others	YYH
18	32.96	C16H22O4	301.1382	−9.4	[M+Na]+, [M+H]+, [M+K]+	149.0216	DBP	Others	NX
19	38.18	C18H30O2	279.2323	1.7	[M+H]+	95.0865	Linolenic acid	Organic acids	HQ
20	46.60	C27H30O16	645.1256	4.4	[M+Cl]−	168.0420	Rutin	Flavonoids	YYH NX HQ
		C27H30O16	633.1444	2.8	[M+Na]+, [M+K]+	207.0296	Rutin	Flavonoids	YYH NX HQ

YYH: Herba Epimedii; NX: Radix Achyranthis Bidentatae; HQ: Radix Astragali; Compared with a reference standard.

3.4. Identification and Analysis of Metabolites in Rat Serum

After the chemical constituents from the GSL get into the body, some components exist in the form of a prototype, but most of them are structurally modified based on the original components, such as oxidation, reduction, and hydration. With the unique data postprocessing function of UNIFI, we analyzed the possible metabolites based on the screened prototype compounds in vivo and finally obtained 42 metabolites in rat serum through artificial screening. After analysis, these 42 metabolites mainly undergo oxidation, hydration, reduction, dehydration, glutathione S-conjugation, and acetylcysteine conjugation reactions based on the prototype compounds. The detailed information of the prototype, metabolite name, molecular weight, and molecular formula is given in Table 3.

Table 3

Identification of the metabolites from GSL in vivo.

NO.	Observed RT (min)	Formula	Observed m/z	Mass error (ppm)	Adducts	Identification
M1	4.8	C39H52O20	875.2792	5.2	[M+Cl]−	Epimedin C + H2 + O
M2	7.4	C28H47N3O9S	646.2978	−5.8	[M+HCOO]−	Linolenic acid + H2O + C10H15N3O6S
M3	7.57	C32H55NO11S	696.3213	3.2	[M+Cl]−	Ecdysterone + H2+H2O + C5H7NO3S
M4	7.72	C27H45O12P	591.2631	9.3	[M−H]−	Ecdysterone + 2x(+O) + HPO3
M5	7.73	C27H47O12P	629.2539	6.4	[M+Cl]−	Ecdysterone + O + H2O + HPO3
M6	7.89	C24H31O9P	493.1677	8.9	[M−H]−	Rubschisantherin − COO + HPO3
M7	8.24	C25H30O10	535.1793	−5.2	[M+HCOO]−	Rubschisantherin + 2x(+O)
M8	8.41	C37H59N3O15S	852.3433	8.4	[M+Cl]−	Ecdysterone + 2x(+O) + C10H15N3O6S
M9	14.8	C24H30O7	429.193	2.7	[M−H]−	Rubschisantherin + O – COO
M10	16.62	C33H36O21	803.1385	−7.2	[M+Cl]−	Rutin − H2O + C6H8O6
M11	19.74	C39H48O19	865.2694	−8.9	[M+HCOO]−	Epimedin A − H2O
M12	19.79	C38H46O20	821.2456	−6.6	[M−H]−	Sagittatoside B + C6H8O6
M13	20.03	C24H30O6	413.199	4.9	[M−H]−	Rubschisantherin − COO
M14	20.04	C38H46O18	835.2618	−5.8	[M + HCOO]−	Epimedin B – H2O
M15	22.34	C27H46O8	497.3131	2.2	[M−H]−	Ecdysterone + H2O
M16	23.09	C33H40O14	659.2317	−4.3	[M−H]−	Icariin + H2 – H2O
M17	24.63	C32H51NO12S	672.312	9.1	[M−H]−	Ecdysterone + 2x(+O) + C5H7NO3S
M18	26.05	C18H34O4	313.2376	−2.9	[M−H]−	Linolenic acid + 2x(+H2O)
M19	27.3	C18H31O6P	409.1519	−8.1	[M+Cl]−	Linolenic acid + O + HPO3
M20	27.48	C27H49O11P	579.2972	5.5	[M−H]−	Ecdysterone + H2 + H2O + HPO3
M21	27.59	C28H49N3O8S	586.3145	−4.1	[M−H]−	Linolenic acid + 2x(+H2) + C10H15N3O6S
M22	27.85	C28H47N3O8S	630.3034	−5.3	[M+HCOO]−	Linolenic acid + H2 + C10H15N3O6S
M23	27.88	C18H30O4	309.2043	−9.1	[M−H]−	Linolenic acid + 2x(+O)
M24	28.66	C27H46O7	527.3212	−2.7	[M+HCOO]−	Ecdysterone + H2
M25	28.82	C27H44O9	557.2997	5.4	[M+HCOO]−	Ecdysterone + 2x(+O)
M26	28.83	C28H47N3O10S	616.2894	−2.7	[M−H]−	Linolenic acid + O + H2O + C10H15N3O6S
M27	28.86	C32H55NO10S	680.3184	−8.6	[M+Cl]−	Ecdysterone + 2x(+H2) + C5H7NO3S
M28	28.97	C32H51NO11S	656.3155	6.9	[M−H]−	Ecdysterone + O + C5H7NO3S
M29	29.08	C27H44O8	531.2733	0.5	[M+Cl]−	Ecdysterone + O
M30	30.24	C18H32O3	295.2255	−8	[M−H]−	Linolenic acid + H2O
M31	31.03	C23H41NO7S	520.2627	7.8	[M+HCOO]−	Linolenic acid + 2x(+H2O) + C5H7NO3S
M32	32.47	C32H47NO8S	640.2711	−0.7	[M+Cl]−	Ecdysterone + 2x(−H2O) + C5H7NO3S
M33	34.78	C18H32O4	347.1975	−5.8	[M+Cl]−	Linolenic acid + O + H2O
M34	34.9	C32H49NO10S	638.2981	−3.6	[M−H]−	Ecdysterone + O – H2O + C5H7NO3S
M35	37.62	C23H30O4	369.2044	−7.4	[M−H]−	Rubschisantherin + 2x(-COO)
M36	38.5	C34H45N3O12S	754.2396	−3	[M+Cl]−	Rubschisantherin-COO + C10H15N3O6S
M37	40.72	C33H40O17	753.2191	−7.5	[M+HCOO]−	Icariin + 2x(+O)
M38	41.15	C33H52O15	733.3252	−5	[M+HCOO]−	Ecdysterone + 2x(+O) + C6H8O6
M39	44.28	C33H56O15	737.3607	0.7	[M+HCOO]−	Ecdysterone + 2x(+H2O) + C6H8O6
M40	51.66	C33H40O23	803.1956	8.5	[M−H]−	Rutin + H2O + C6H8O6
M41	53.66	C39H51O21P	921.2341	−1.5	[M+Cl]−	Epimedin C + H2–H2O + HPO3
M42	53.67	C37H47N3O22S	916.2292	−0.9	[M−H]−	Rutin + H2 + C10H15N3O6S

+H2: reduction; +O: oxidation; +H2O: hydration; +HPO3: phosphorylation; +C6H8O6: glucuronidation; +C5H7NO3S: acetyl cysteine conjugation; +C10H15N3O6S: glutathione S-conjugation; –H2O: dehydration; –COO: decarboxylation.

3.5. Analysis of GSL by UPLC-Q-TOF-MS

3.5.1. Flavonoids

Flavonoids and their glycosides are the main ingredients and the major bioactive components of GSL. In this study, 3 flavones, 41 flavonols, 5 isoflavones, 1 isoflavanone, and 1 flavanol were determined by the matching of the mass spectrometry data with the UNIFI analysis platform.

For flavonoid glycosides, the glycosidic bonds connected by oxygen atoms could be cleaved in both positive and negative ion modes, and most of them were characterized by neutral losses such as 162 Da (Glc) and 146 Da (Rha) [24]. It was difficult to directly remove the glycosyl groups connected by carbon-glycosidic bonds and often produce [M+H−120]+ fragment [25]. As we all know, the main cleavage behavior of flavonoid aglycones was the retro Diels–Alder reaction (RDA) cleavage pathway and the loss of free radicals and/or small molecules (such as CH3, CO, and CO2) [26]. By comparing the retention time and fragmentation patterns with standard substance, peaks 14, 31, 33, 34, 35, 37, and 41 in Figure 1 were exactly identified as calycosin-7-glucoside, ononin, calycosin, epimedin A, epimedin B, epimedin C, and icariin, respectively. Here, we took epimedin C and icariin as examples to depict the fragmentation mode of these ingredients.

Epimedin C showed quasi-molecular ion [M+H]+ at m/z 823.3014 in positive ion mode and yielded fragment ions at m/z 531.1852 and 515.1931 by losses of 2 molecules of rhamnose and a molecule of ORha-Rha group, respectively. Then, the ion at m/z 515.1931 loses a molecule of Glc to generate an ion at m/z 369.1322, and the ion at m/z 369.1322 further loses a functional group of C4H8 to produce ion at m/z 313.0699. The mass spectrogram and possible fragmentation pathways of epimedin C in positive ion mode are shown in Figure 3.

Figure 3

The mass spectrogram and fragmentation pathways of epimedin C in positive ion mode.

In the positive ion mode, the mass-to-charge ratio of the quasi-molecular ion peak of compound 41 was 677.2433 [M+H]+ as shown in Table 1. The mass-to-charge ratio of the fragment ions produced by the precursor ions were 531.1851 [M+H−Rha]+, 369.1321 [M+H−Glc]+, and 313.0698 [M+H−C4H8]+, which are consistent with those of icariin. The mass spectrogram and potential fragmentation pathways of icariin in positive ion mode are shown in Figure S3.

3.5.2. Terpenoids

The terpenoids in GSL primarily included monocyclic monoterpenoids, cycloartane-type tetracyclic triterpenoids, and oleanane-type pentacyclic triterpenoids. Among them, the number of the above categories in GSL was 3, 4, and 11, respectively.

Triterpene saponins in GSL mainly exist in the form of aglycones binding with sugars, such as glucose, rhamnose, and xylose. In mass spectrometric analysis, triterpenoid saponins are mostly in the form of de-sugar or continuous de-sugar fragment ions [27]. Such compounds also had the loss of CO2, COOH, CH2OH, and other complex groups. Compound 49 gave a deprotonated molecule [M−H]− at m/z 1117.5026 and produced predominant fragment ions at m/z 997.4956 [M−H−COOH−CH2OH−CO2]− and m/z 631.2021 [M−H−C2H2O−2Glc]− in negative ion mode (Figure 4). It was consistent with previous literature [28] and was finally identified as achyranthoside D. Peak 51 was identified clearly as ginsenoside Ro with a standard substance, and its mass fragmentation pattern is demonstrated in detail (Figure S4). In the negative ion mode, ginsenoside Ro gave [M−H]− ion at m/z 955.4884, along with two major fragment ions at m/z 793.4236 [M−H−Glc]− and 631.2021[M−H−2Glc]− in mass spectrometry under high-energy conditions. From the cleavage pathway of these triterpenoid saponins, they tend to lose the sugar group at the C28 position first under the action of high energy of MSE. The possible reason for this phenomenon is that the ester bond at the C28 position is easier to break than the ether bond at the C3 position.

Figure 4

The mass spectrogram and fragmentation pathways of achyranthoside D in negative ion mode.

3.5.3. Phenylpropanoids

Six phenylpropanoids were recognized as the major active ingredients in GSL. Among them, a total of 2 phenylpropionic acids, 1 phenylpropanol, 1 styrene, and 2 lignans were identified. Simple phenylpropanoids belong to phenylpropane derivatives in structure and exist in plants in the form of glycosides or esters, which can be combined with sugars and polyols. In mass spectrometric analysis, phenylpropanoids are mainly manifested by the loss of sugar, neutral loss, and loss of other complex groups [29]. Compound 11 showed a deprotonated molecule [M−H]− at m/z 539.2149 and produced predominant fragment ions at m/z 491.1857 [M−H−CH2O−H2O]−, m/z 479.0789 [M−H−C2H4O2]−, and m/z 317.0238 [M−H−C3H6O−Rha−OH]− in negative ion mode. By confirmation of fragment ions, we preliminarily identified compound 11 as icariside E1. In the case of compound 44, it generated a base peak ion at m/z 481.1785 [M+Na]+ in positive ion mode, along with a major fragment ion at m/z 387.1427 [M+Na−C4H8O]+, which was consistent with a previous study [30]. Finally, it was assigned to be rubschisantherin. The detailed mass spectrogram and fragmentation pathways are shown in Figures 5 and S5.

Figure 5

The mass spectrogram and fragmentation pathways of icariside E1 in negative ion mode.

3.5.4. Steroids

In the UNIFI results interface, 5 steroids were automatically matched. The cleavage of steroids and their aglycones is complicated. Besides RDA cleavage, dehydration, and demethylation of the hydroxyl group, the side chain at position 17 often falls off. Peak 18 was ascertained to be ecdysterone by contrast with reference standards. As shown in Figure 6, ecdysterone displayed a hydrogenated ion at m/z 481.3134 [M+H]+ with a molecular formula C27H44O7 and lost H2O to generate an ion at m/z 445.2923 [M+H−2H2O]+. Further loss of the C4H10O group resulted in fragmentation with a m/z of 371.2244 [M+H−2H2O−C4H10O]+.

Figure 6

The mass spectrogram and fragmentation pathways of ecdysterone in positive ion mode.

3.5.5. Others

Some compounds with fewer species and lower concentrations are assigned to this category. The mass spectra data extracted from MassLynx workstation were matched with UNIFI software, and the results were verified by literature analysis [31]. A total of 13 compounds were inferred, including anthraquinones, glycosides, organic acids, and others. Specific mass spectrometry data are listed in Table 1.

4. Conclusions

In this experiment, UPLC-Q-TOF-MS technology combined with UNIFI software was used for the first time to comprehensively and systematically analyze the in vitro and in vivo chemical constituents of GSL. We summarized the cleavage law of flavonoids, triterpene saponins, phenylpropanoids, and steroids in the mass spectrum and initially explored the prototype compounds and metabolites of GSL in rat serum. These results provide a technical basis for the comprehensive and effective quality control and pharmacodynamic material basis of GSL. In addition, some chromatographic peaks with better response in GSL are unknown ingredients, which deserve further study.