Identification of Metabolites of Eupatorin in Vivo and in Vitro Based on UHPLC-Q-TOF-MS/MS

Eupatorin is the major bioactive component of Java tea (Orthosiphon stamineus), exhibiting strong anticancer and anti-inflammatory activities. However, no research on the metabolism of eupatorin has been reported to date. In the present study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) combined with an efficient online data acquisition and a multiple data processing method were developed for metabolite identification in vivo (rat plasma, bile, urine and feces) and in vitro (rat liver microsomes and intestinal flora). A total of 51 metabolites in vivo, 60 metabolites in vitro were structurally characterized. The loss of CH2, CH2O, O, CO, oxidation, methylation, glucuronidation, sulfate conjugation, N-acetylation, hydrogenation, ketone formation, glycine conjugation, glutamine conjugation and glucose conjugation were the main metabolic pathways of eupatorin. This was the first identification of metabolites of eupatorin in vivo and in vitro and it will provide reference and valuable evidence for further development of new pharmaceuticals and pharmacological mechanisms.


Introduction
Eupatorin (5,3 -di-hydroxy-6,7,4 -tri-methoxy-flavone, Figure 1), belonging to the natural methoxyflavone compound, is widely found in Java tea (Orthosiphon stamineus, OS) which is a popular medicinal herb used in traditional Chinese medicine as a diuretic agent and for renal system disorders in Southeast Asia and European countries [1][2][3]. OS has gained a great interest nowadays due to its wide range of pharmacological effects such as antibacterial, antioxidant, hepatoprotection, antidiabetic, anti-hypertension, anti-inflammatory and antiproliferative activities [4][5][6][7][8][9]. Eupatorin, as a major bioactive flavonoid constituent in OS possesses numerous strong biological activities, including anticancer, anti-inflammatory and vasorelaxation activities [10][11][12][13][14][15][16][17]. Its anticancer activities have attracted more and more attention and it was expected to be developed as a cancer chemopreventive and as an adjuvant chemotherapeutic agent. Although there is literature on the qualitative and quantification profile of eupatorin in OS [6], the metabolism study of eupatorin has not been studied to date, which was necessary for the exploration of the biological activity and the clinical therapeutic effect of eupatorin. Thus, an investigation is essential to explore the identification of metabolites of eupatorin for further understanding of its biological activities. To the best of our knowledge, a series of biotransformations will occur when drugs are orally taken into the body, there are four aspects of pharmacological consequences in these biotransformation processes: (1) Transforming into inactive substances; (2) transforming the drug with no pharmacological activity into active metabolites; (3) changing the types of pharmacological actions of drugs; (4) and producing toxic substances [18]. Therefore, it is extremely crucial to study the metabolism of drugs in vivo to make sure of safety of use. In addition, as the main metabolic organ of the human body, the liver is rich in enzymes, especially cytochrome P450 enzymes, which are closely related to the biological transformation of drugs [19]. Furthermore, the gastrointestinal tract is also a vital place for drug metabolism, and its intestinal flora have a significant impact on drug absorption, metabolism and toxicology [20,21]. Hence, in this paper, mass spectrometry was employed to investigate the metabolism of eupatorin in rats, liver microsomes and intestinal flora, in order to characterize the metabolites and structural information of the products, which will lay a foundation for further studies on the safety and efficacy of metabolites and will provide greater possibilities for the development of new drugs.
With the development of technology, a quadrupole time-of-flight mass spectrometry has been widely used as a reliable analytical technique to detect metabolites due to its advantages of high resolution, high sensitivity, high-efficiency separation and accurate quality measurement [22,23]. In this study, high-sensitivity ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) full scan mode, electrospray ionization (ESI) source negative ion mode monitoring combined with multiple mass loss (MMDF) and dynamic background subtraction (DBS) were employed to collect data online. Correspondingly, multiple data processing methods were applied by using PeakView 2.0 and MetabolitePilot 2.0.4 software developed by AB SCIEX company, including a variety of data handing functions such as the extraction of ion chromatograms (XIC), mass defect filter (MDF), product ion filter (PIF) and neutral loss filtering (NLF), which provided accurate secondary mass spectral information [24]. Based on the above methods, the metabolic pathways of eupatorin were explored and summarized for the first time and 51 metabolites in vivo and 60 metabolites in vitro were finally identified. These metabolic studies are important parts of drug discovery and development and can also provide a basis for further pharmacological research.

Analytical Strategy
In this study, UHPLC-Q-TOF-MS/MS combined with an online data acquisition and multifarious processing methods was adopted to systematically identify the metabolites of eupatorin in vivo and in vitro.
The workflow of the analytic procedure was segmented into three steps. First, an online fullscan data acquisition was performed based on the MMDF and DBS to collect data online and to capture all potential metabolites. Next, a multiple data processing method was employed by using PeakView 2.0 and MetabolitePilot 2.0.4 software, which contained many data-processing tools such as XIC, MDF, PIF and NIF, these provided accurate MS/MS information to determine the metabolites of eupatorin. Finally, plenty of metabolites were identified according to accurate mass datasets, specific secondary mass spectrometry information and so on. With regard to the isomers of metabolites, Clog P values calculated by ChemDraw 14.0 were used to further distinguish them. To the best of our knowledge, a series of biotransformations will occur when drugs are orally taken into the body, there are four aspects of pharmacological consequences in these biotransformation processes: (1) Transforming into inactive substances; (2) transforming the drug with no pharmacological activity into active metabolites; (3) changing the types of pharmacological actions of drugs; (4) and producing toxic substances [18]. Therefore, it is extremely crucial to study the metabolism of drugs in vivo to make sure of safety of use. In addition, as the main metabolic organ of the human body, the liver is rich in enzymes, especially cytochrome P450 enzymes, which are closely related to the biological transformation of drugs [19]. Furthermore, the gastrointestinal tract is also a vital place for drug metabolism, and its intestinal flora have a significant impact on drug absorption, metabolism and toxicology [20,21]. Hence, in this paper, mass spectrometry was employed to investigate the metabolism of eupatorin in rats, liver microsomes and intestinal flora, in order to characterize the metabolites and structural information of the products, which will lay a foundation for further studies on the safety and efficacy of metabolites and will provide greater possibilities for the development of new drugs.
With the development of technology, a quadrupole time-of-flight mass spectrometry has been widely used as a reliable analytical technique to detect metabolites due to its advantages of high resolution, high sensitivity, high-efficiency separation and accurate quality measurement [22,23]. In this study, high-sensitivity ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) full scan mode, electrospray ionization (ESI) source negative ion mode monitoring combined with multiple mass loss (MMDF) and dynamic background subtraction (DBS) were employed to collect data online. Correspondingly, multiple data processing methods were applied by using PeakView 2.0 and MetabolitePilot 2.0.4 software developed by AB SCIEX company, including a variety of data handing functions such as the extraction of ion chromatograms (XIC), mass defect filter (MDF), product ion filter (PIF) and neutral loss filtering (NLF), which provided accurate secondary mass spectral information [24]. Based on the above methods, the metabolic pathways of eupatorin were explored and summarized for the first time and 51 metabolites in vivo and 60 metabolites in vitro were finally identified. These metabolic studies are important parts of drug discovery and development and can also provide a basis for further pharmacological research.

Analytical Strategy
In this study, UHPLC-Q-TOF-MS/MS combined with an online data acquisition and multifarious processing methods was adopted to systematically identify the metabolites of eupatorin in vivo and in vitro.
The workflow of the analytic procedure was segmented into three steps. First, an online full-scan data acquisition was performed based on the MMDF and DBS to collect data online and to capture all potential metabolites. Next, a multiple data processing method was employed by using PeakView 2.0 and MetabolitePilot 2.0.4 software, which contained many data-processing tools such as XIC, MDF, PIF and NIF, these provided accurate MS/MS information to determine the metabolites of eupatorin. Finally, plenty of metabolites were identified according to accurate mass datasets, specific secondary mass spectrometry information and so on. With regard to the isomers of metabolites, Clog P values calculated by ChemDraw 14.0 were used to further distinguish them. Generally speaking, the larger the Clog P value, the longer the retention time will be in the reversed-phase chromatography system [25][26][27].

Mass Fragmentation Behavior of Eupatorin
In order to identify the metabolites of eupatorin, it is of significance to understand the pyrolysis of parent drug (M0). The chromatographic and mass spectrometric behaviors of eupatorin were explored in the negative ESI scan mode by UHPLC-Q-TOF-MS. Eupatorin (C 18  O 2 , respectively. The product ion at m/z 285.0398 was created by dropping CO from the ion at m/z 313.0348. Last but not the least, the conspicuous product ion at m/z 147.0461 was formed because of the Retro-Diels-Alder (RDA) reaction in ring C of the flavonoid, which gained the ion at m/z 132.0214 by loss of CH 3 [28]. The MS/MS spectrum and the fragmentation pathways of eupatorin are shown in Figure 2. Generally speaking, the larger the Clog P value, the longer the retention time will be in the reversedphase chromatography system [25][26][27].

Mass Fragmentation Behavior of Eupatorin
In order to identify the metabolites of eupatorin, it is of significance to understand the pyrolysis of parent drug (M0). The chromatographic and mass spectrometric behaviors of eupatorin were explored in the negative ESI scan mode by UHPLC-Q-TOF-MS. Eupatorin (C18H16O7) was eluted at 12.22  .0434 by loss of CO2 and 2O, C4H6O3, C7H6O2, respectively. The product ion at m/z 285.0398 was created by dropping CO from the ion at m/z 313.0348. Last but not the least, the conspicuous product ion at m/z 147.0461 was formed because of the Retro-Diels-Alder (RDA) reaction in ring C of the flavonoid, which gained the ion at m/z 132.0214 by loss of CH3 [28]. The MS/MS spectrum and the fragmentation pathways of eupatorin are shown in Figure 2.

Identification of Metabolites in Vivo and in Vitro
Metabolites M1, M2 and M3 (C 17 H 14 O 7 ) were isomers with the deprotonated molecular ions [M-H] − at m/z 329.0660, 329.0668 and 329.0662, which were 14 Da (CH 2 ) lower than that of M0. They were eluted at 9.93 min, 10.27 min and 10.79 min, respectively. In the MS/MS spectrum, product ions at m/z 314.0427, 313.0384, 299.0188 and 285.0371 were formed after losing CH 3 , O, 2CH 3 and CO 2 , respectively. The prominent fragment ion at m/z 133.0287 created after the RDA reaction was 14 Da lower than the ion m/z 147.0461 of the parent drug, suggested that CH 2 was lost at the methoxy group at 4 position. At the same time, the fragment ions at m/z 207.7129 and 207.7166 were 14 Da lower than that of M0, which showed that the loss of CH 2 occurred at the methoxy group at 6 or 7 position of A ring. Additionally, the Clog P values of M1, M2 and M3 were 2.26422, 2.26434, 2.51422, respectively. Therefore, M1-M3 were illustrated according to the above information.
Metabolites M4 and M5 (C 16 H 12 O 7 ) were eluted at 7.26 and 8.50 min, with the deprotonated molecular ions [M-H] − at m/z 315.0500 and 315.0504, 28 Da (C 2 H 4 ) lower than that of the parent drug, which indicated that it lost 2CH 2 . Fragment ions at m/z 300.0279 and 297.1740 were generated by loss of CH 3 and H 2 O, respectively. The product ion at m/z 269.1760 was obtained through dropping CO from the ion at m/z 297.1740. According to the dominant fragment ion at m/z 133.0270 gained by the RDA reaction, loss of CH 2 and CH 2 occurred at the position of 4 , 6 or 4 , 7. In addition, the distinctive ion at m/z 147.0821 was similar with that of the parent drug, which implied that the reaction occurred at the position of 6 and 7.
Metabolite M6 (C 17 H 14 O 6 ) was obtained with a peak at m/z 313.0713 in the UPLC system, which was eluted at 13.86 min, 30 Da (CH 2 O) lower than that of eupatorin. Prominent fragment ions at m/z 298.0483 and 283.0250 were created by dropping CH 3 and CH 3 successively. In addition, the characteristic fragment ions at m/z 117.0364 was produced by RDA reaction, which was 30 Da lower than that of M0, showing that loss of CH 2 O occurred at the position of 4 . Similarly, the product ion at m/z 147.0078 was consistent with M0, indicating that loss of CH 2 O occurred at the position of 6 or 7. Thus, it was speculated that it may have three missing CH 2 O sites.
Metabolite M7 (C 16 H 12 O 6 ) was detected at 10.10 min and exhibited the molecular ion [M-H] − at m/z 299.0562, which was 44 Da lower than that of M0. Based on the information of chemical elements and software provided, it indicated that M7 lost CH 2 O and CH 2 . Crucial fragment ions at m/z 284.0326 and 251.1281 were obtained by loss of CH 3 and 3O from M7, respectively. Furthermore, M7 had common fragment ion at m/z 146.9687 with that of the parent drug, it is equally important that the noteworthy fragment ion at m/z 281.1787 was generated by loss of H 2 O from M7, which implied that loss of CH 2 O and CH 2 occurred at the position of 7 or 6, respectively. Hence, it was identified.
Metabolite M8 (C 16   Metabolite M18 (C 18 H 16 O 9 ), the deprotonated molecular ion of m/z 375.0709 was observed at the retention time of 9.90 min, which was 32 Da (2O) higher than that of eupatorin. A series of product ions at m/z 329.0669, 221.1216 and 178.9947 were detected by loss of CH 2 O 2 , C 7 H 6 O 4 and RDA reaction in its secondary mass spectrum. Product ions at m/z 314.0434 and 299.0191 were produced by losing CH 3 and CH 3 continuously from the ion at m/z 329.0669. What's more, the key fragment ions at m/z 178.9947 was 32 Da higher than 147.0461 of eupatorin, implying that di-oxidation reaction occurred in the B ring, then M18 was identified.
Metabolite were obtained in the extracted chromatogram at m/z 357.0972 and 357.0969 with the retention time of 10.02 min and 12.86 min, which were 14 Da (CH 2 ) higher than that of eupatorin. The diagnostic fragment ions at m/z 342.0740, 327.0503, 312.0266 and 297.0033 were attributed to the loss of CH 3 successively. In addition, because of the prominent fragment ions at m/z 235.0434 and 147.0433 obtained after RDA reaction, it was proposed that methylation happened at hydroxyl group at 5 position. Nevertheless, the fragment ion at m/z 161.0269 was 14 Da higher than 147.0461 of eupatorin, indicating that it occurred at C-3 of B ring. Furthermore, the Clog P values of M22 and M23 were 2.06632 and 3.18323, respectively, so they were verified.
Metabolites 14 min, respectively, which were 176 Da higher than that of eupatorin, suggesting that glucuronidation was carried out. The key product ion at m/z 343.0822 was yielded by dropping a glucuronic acid. Moreover, the crucial ion at m/z 146.9662 was similar to the fragment ion at m/z 147.0461 and while the fragment ion at m/z 397.0442 was 176 Da higher than that of the parent drug, indicating that glucuronidation happened at the hydroxyl group at 5 position. Nevertheless, the prominent fragment ions at m/z 323.0173 was 176 Da larger than 147.0461 of M0, inferring that the reaction occurred at the hydroxyl group at 3 position. Furthermore, M28 and M29 were also proved by the different Clog P values of −0.494983 and 0.621934, respectively.
Metabolite   18 Da higher than that of M14-M17, implying that M67 might undergo oxidation followed by internal hydrolysis. In the MS/MS spectrum of M67, the representative product ion at m/z 181.0136 tested after RDA reaction was 34 larger than 147.0461 of eupatorin, while the fragment ion at m/z 239.0435 was 18 times higher than that of eupatorin, so internal hydrolysis happened at C-2 and C-3 and oxidation is most likely to occur at C-5 [29].
Metabolite M68 (C 19 H 17 NO 8 ) was observed with a peak at m/z 386.0865 in the UPLC system, which was eluted at 10.00 min, 57 Da higher than that of M1-M3. The fragment ion at m/z 329.0662 was acquired, corresponding to the loss of glycine. Additionally, the conspicuous fragment ions at m/z 264.1192 and 147.0973 were yielded through the loss of C 7 H 6 O 2 and RDA reaction, implying that the loss of CH 2 and glycine conjugation were connected to A ring. Hence, there were two possible metabolites of M68.
Metabolite The detected metabolites are listed in Table 1. Moreover, their XICs are exhibited in Figure 3.

Metabolic Pathways of Eupatorin
The metabolites of eupatorin in rats after oral administration, in liver microsomes and intestinal flora through incubation was identified in this study. As a result, a total of 51 metabolites in vivo were detected, including 8 metabolites in plasma, 5 metabolites in bile, 36 metabolites in urine and 32 metabolites in feces. Meanwhile, 60 metabolites in vitro were observed, including 22 metabolites in liver microsomes and 53 metabolites in intestinal flora. The proposed metabolic pathways of

Metabolic Pathways of Eupatorin
The metabolites of eupatorin in rats after oral administration, in liver microsomes and intestinal flora through incubation was identified in this study. As a result, a total of 51 metabolites in vivo were detected, including 8 metabolites in plasma, 5 metabolites in bile, 36 metabolites in urine and 32 metabolites in feces. Meanwhile, 60 metabolites in vitro were observed, including 22 metabolites in liver microsomes and 53 metabolites in intestinal flora. The proposed metabolic pathways of eupatorin in vivo, in rat liver microsomes and in rat intestinal flora were shown in Figure 4. It is worth mentioning that the loss of CH 2 , CH 2 O, O, oxidation, glucuronidation and ketone formation was the primary metabolic step that produced further reactions such as sulfate conjugation, hydrogenation, N-acetylation, methylation, demethylation, internal hydrolysis, glycine conjugation, glutamine conjugation and glucose conjugation. Moreover, all metabolic changes above had taken place in vivo and in vitro. However, glycine conjugation was just present in vivo, while glutamine conjugation and glucose conjugation merely existed in vitro.

Comparison of Metabolites in Vivo and in Vitro
Drug metabolism plays a significant impact on various fields of pharmaceutical mechanisms as well as drug development and clinical use. In this work, the metabolism of eupatorin in vivo (plasma, bile, urine and feces) and in vitro (rat liver microsomes and intestinal flora) was investigated. In vivo; rat urine and feces possessed high activity for eupatorin metabolism, which were identified as having 36 and 32 metabolites, respectively. Nevertheless, only 8 metabolites were observed in rat plasma and 5 metabolites were detected in rat bile, suggesting that the rat plasma and bile might hold low biotransformation activity [30]. In vitro, 53 metabolites were obtained in rat intestinal flora while 22 metabolites were identified in rat liver microsomes, which implied that most metabolites could be excreted in intestinal flora samples and intestinal tract was more suitable for rapid identification of metabolites of eupatorin in vitro, with enormous catalytic and metabolic capacity which exceeds that of the liver microsomes [24]. Thus, the intestinal tract is considered as an extremely vital organ in the biotransformation of eupatorin.  Figure 4. It is worth mentioning that the loss of CH2, CH2O, O, oxidation, glucuronidation and ketone formation was the primary metabolic step that produced further reactions such as sulfate conjugation, hydrogenation, N-acetylation, methylation, demethylation, internal hydrolysis, glycine conjugation, glutamine conjugation and glucose conjugation. Moreover, all metabolic changes above had taken place in vivo and in vitro. However, glycine conjugation was just present in vivo, while glutamine conjugation and glucose conjugation merely existed in vitro. (G1)

Comparison of Metabolites in Vivo and in Vitro
Drug metabolism plays a significant impact on various fields of pharmaceutical mechanisms as well as drug development and clinical use. In this work, the metabolism of eupatorin in vivo (plasma, bile, urine and feces) and in vitro (rat liver microsomes and intestinal flora) was investigated. In vivo;

Metabolite Activity of Eupatorin
It has been reported in the literature that OS was taken as a beverage to improve health and for treatment of kidney disease, bladder inflammation and urethritis [1,2]. As its major active ingredient, eupatorin has also been reported to have meaningful anti-inflammatory activity [15,16]. In this study, the metabolites of eupatorin in urine samples were the largest, which may be related to the therapeutic effects of cystitis, nephritis and urethritis. In addition, many of the metabolites of eupatorin have been studied. For example, M4a namely nepetin, is a natural flavonoid present in different plants. In recent years, accumulating evidence has shown that nepetin exhibits various pharmacological activities, especially potent anti-inflammatory properties, which might be related to the strong anti-inflammatory activity of eupatorin [30][31][32]. Overall, the identification of metabolites of eupatorin provides a basis for new pharmacological studies and these metabolites will be further explored in the future.

Animals and Drug Administration
Eighteen male Sprague-Dawley (SD) rats (220-220 g, 12-14 weeks old) were purchased from the Experimental Animal Research Center of Hebei Medical University (Certificate No.1811164). The conditions of temperature (22-25 • C), humidity (55-60%) and light (12 h light/dark cycle) were standard for 7 days before being used. All rats were fasted for 12 h but allowed water before the experiments. These rats were divided into six groups randomly with three rats per group. Groups 1, 3 and 5 were the control groups for blank blood, blank bile, blank urine and feces, respectively. Groups 2, 4 and 6 were the drug groups for blood, bile, urine and feces, respectively. Rats in groups 2, 4, 6 were given eupatorin by gavage, which dissolved in a 0.5% CMC-Na solution at a dose of 50 mg/kg. Nevertheless, an equal 0.5% CMC-Na solution without eupatorin was orally given to groups 1, 3, 5.
All rat experiments were conducted in accordance with the committee's guidelines on the Care and Use of Laboratory Animals.

Bio-Sample Collection
The plasma samples collection: About 300 µL-500 µL for each blood sample was gathered from the eye canthus of rat into 1.5 mL heparinized tubes at 0.083, 0.167, 0.25, 0.5, 1, 2, 3, 6, 9, 12 and 24 h after gavage. Every blood sample were centrifuged immediately at 1920 g for 10 min at 4 • C to collect the supernatant. After that all collected plasma samples were combined and stored at −80 • C.
The urine and feces collection: The rats were separately housed in metabolic cages with free access to deionized water to collect the urine and feces samples over a 0-72 h period after gavage [35,36]. Finally, all the urine and feces samples were separately mixed, and they were placed at −80 • C before pretreatment was conducted.

Bio-Sample Pretreatment
All biological samples were disposed with two methods: Protein precipitation and liquid-liquid extraction were performed on the combined plasma, bile and urine with three times of methanol and ethyl acetate, respectively. Next, the mixture was vortexed for 5 min and centrifuged at 21,380× g for 10 min at 4 • C to obtain the supernatant, which was then collected and dried under nitrogen flow.
Dried and powdered feces samples were severally added to 3-fold methanol and ethyl acetate and then were ultrasonically extracted for 45 min. After centrifugation for 10 min at 21,380× g, they were dried under nitrogen gas like the supernatant in plasma, bile and urine samples.
150 µL methanol was added to the residua above with an ultrasonic operation for 15 min, centrifugation at 21,380× g for 10 min to gain the supernatant which were ultimately passed through the 0.22 µm millipore filters before injecting into the chromatographic system for further analysis. The control group was handled the same as the drug group.

Phase I Metabolism
The typical incubation mixture was carried out in a PBS buffer (pH 7.4) with a final volume of 200 µL, which consisted of liver microsomal protein (1.0 mg/mL), eupatorin (100 µmol/L), MgCl 2 (3.3 mmol/L), and β-NADPH (1.3 mmol/L) [37]. Preincubation was conducted at 37 • C for 5 min, subsequently NADPH was added to start the reaction. After incubation at 37 • C for 90 min, the reaction was terminated by adding 1 mL of ethyl acetate. Next, vortex and centrifugation for 5 and 10 min, respectively, and then the organic phase was gathered and evaporated under nitrogen gas. 100 µL of acetonitrile was put in the residua and they were eventually passed through the 0.22 µm millipore filters and placed at −20 • C before analysis. Groups contained blank groups incubated without the addition of eupatorin, the control groups incubated without the addition of NADPH and the sample groups, which were implemented in triplicate with the same treatment [38,39].

Phase II Metabolism
The representative incubation mixture was performed in a PBS buffer (pH 7.4) with a final volume of 200 µL, which including liver microsomal protein (1.0 mg/mL), eupatorin (100 µmol/L), MgCl 2 (3.3 mmol/L), and UDPGA (2 mmol/L). Preincubation was implemented at 37 • C for 20 min, subsequently UDPGA was added to begin the reaction. After incubation at 37 • C for 1 h, the reaction was ceased by adding 200 µL of ice-acetonitrile. Next, vortex and centrifugation for 5 and 10 min, respectively. In addition, the supernatant was passed through the 0.22 µm millipore filter before injecting into the UHPLC-Q-TOF-MS/MS system for analysis. Groups contained blank groups incubated without the addition of eupatorin, the control groups incubated without the addition of UDPGA and the sample groups, which were carried out in triplicate with the same treatment.

Preparation of Intestinal Flora Culture Solution
Fresh intestinal contents (3 g) taken from SD rats were combined with anaerobic culture medium (30 mL) instantly. After stirring with a glass rod, filtered with gauze to obtain the intestinal bacterial liquid.

Sample Preparation
Eupatorin (1 mg/ mL,100 µL) was added to intestinal flora culture medium (1 mL), which was then filled with nitrogen without oxygen. The reactions were terminated by adding 3 volumes of methanol after incubation for 12 h. Next, the mixtures were vortexed for 5 min and centrifuged for 10 min at 21,380 g. Subsequently, the organic phases were collected and evaporated under nitrogen gas, and 100 µL of methanol was added to the residua, vortexed and centrifuged again for 5 and 10 min, respectively. Before analysis, the supernatant was passed through the 0.22 µm millipore filter. Blank groups were incubated without eupatorin, meanwhile the control groups were incubated not in intestinal flora culture solution but in anaerobic culture medium, but others were the same.

Conclusions
In conclusion, the identification of metabolites of eupatorin in vivo and in vitro had achieved great success firstly by means of UHPLC-Q-TOF-MS/MS combined with a powerful and efficient data acquisition and processing method. The results displayed that a total of 71 metabolites were characterized: 51 metabolites were identified in vivo (8 metabolites in the plasma, 5 metabolites in the bile, 36 metabolites in the urine and 32 metabolites in the feces), while 60 metabolites were detected in vitro (22 metabolites in the rat liver microsomes and 53 metabolites in rat intestinal flora). This study was expected to benefit future efficacy and safety studies on eupatorin and provide guidelines for intake of OS. There is no doubt that further studies are needed to confirm the impact of these metabolites on human health and safety, thus providing reasonable recommendations for the consumption of foods and drugs containing eupatorin.