Identification of Metabolites of Aurantio-Obtusin in Rats Using Ultra-High-Performance Liquid Chromatography-Q-Exactive Orbitrap Mass Spectrometry with Parallel Reaction Monitoring

Aurantio-obtusin (AO) is a major anthraquinone compound isolated from Cassiae Semen or Duhaldea nervosa, which possesses diverse pharmacological effects. Previous studies have shown that it has a good effect on lowering blood lipids and treating various diseases. A few studies have also reported about its metabolites. A rapid and reliable method using ultra-high-performance liquid chromatography-Q-Exactive Orbitrap mass spectrometry and multiple data-processing technologies was established to investigate the metabolites of AO in the plasma and various tissues of rats, including the heart, liver, spleen, lung, kidneys, and brain. Finally, a total of 36 metabolites were identified in the plasma of rats, which could be very beneficial for understanding the effective form of AO metabolites leading to new drug discovery. The result demonstrated that this strategy, especially parallel reaction monitoring, has shown a wide range of applications in the identification of metabolites.


Introduction
Aurantio-obtusin (AO) is a lipophilic anthraquinone compound, which is isolated from traditional Chinese medicine such as Cassiae Semen and Duhaldea nervosa [1,2]. ey are both edible and medicinal plants, which have been used for the treatment of hyperlipidemia, hypertensive and rheumatoid arthritis, etc. [3][4][5][6]. AO possesses a variety of biological activities, such as antihypertensive activity, antiallergic responses, and anti-inflammatory activity [7,8]. In recent years, research on AO has been increasing, focusing on the pharmaceutical analysis in vitro and pharmacological studies [8,9]; however, a few in vivo investigations have also been done, especially on the metabolites of AO in rats. A total of 21 metabolites of AO were identified in plasma [10,11]. erefore, it is of great significance to investigate the metabolism of AO in vivo.
Nowadays, ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry has become one of the essential techniques for the detection and characterization of metabolites, especially for the identification of metabolites in trace amounts or in complex samples [12][13][14][15]. Generally, the full scan with data dependence MS 2 was adopted for the MS n acquiring of the sample. However, the MS 2 of the metabolites in trace amounts could not be generated in this mode because the MS 2 will not be triggered if the abundance of the ions in MS 1 was not reached to the top of 3 [16,17]. us, the metabolites were not completely investigated due to the limitations of the analytical method. In recent years, parallel reaction monitoring (PRM) scanning mode has been developed for achieving the designed ion's MS 2 data [18][19][20]. erefore, a systematic analytical strategy for the metabolites of AO was proposed based on UHPLC Q Exactive Orbitrap with PRM scanning mode. Finally, a total of 36 metabolites were identified in the plasma of rats, which will be very beneficial for understanding the effective form of AO and new drug discovery. e result demonstrated that this strategy, especially parallel reaction monitoring, has shown the ability of a wide range of applications in the identification of metabolites. e rats were housed in a controlled room at standard temperature (24 ± 2°C) and humidity (70 ± 5%) for a week. During this time, free access to food and water was provided to the rats to adapt to the environment. en, the rats were randomly divided into the drug group and the blank group to assess the plasma and various tissues, including heart, liver, spleen, lung, kidney, and brain. e rats were fasted for 12 h with free access to water prior to the experiment. e animal protocols were approved by the Medicine Ethics Review Committee for Animal Experiments at the Hunan University of Medicine. e animal facilities and protocols complied with the Guide for the Care and Use of Laboratory Animals.

Drug Administration and Collection of Biological Samples.
AO was dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na) solution and orally administrated to rats in the drug group at a dose of 200 mg/kg, while 0.5% CMC-Na aqueous solution (2 mL) was administered to rats in the blank group. 0.5 mL of the blood samples was collected from the external jugular vein after the rats were given the drug orally and kept in heparinized tubes for 0.5, 1, 2, and 4 h, and then the plasma was obtained by centrifuging the tubes at 3000 rpm for 15 min at 4°C, respectively. e organs, including heart, liver, spleen, lung, kidney, and brain, were, respectively, harvested from rats in drug and blank groups at 4 h after dosing and washed with cold biological saline. All of the samples were stored at −80°C before further sample pretreatment.

Sample Preparation.
In order to detect a maximum number of metabolites of AO, three preparation methods were performed in this study. e first method was processed by solid-phase extraction (SPE). e SPE cartridges were activated and equilibrated by eluting with methanol (5 mL) and deionized water (5 mL) successively. en, the plasma sample (0.1 mL) was loaded on the SPE column, followed by flushing with deionized water (3 mL) and methanol (3 mL). Afterward, the methanol eluted was collected and dried under the stream of nitrogen at room temperature to gain the residues, which were reconstituted in 0.1 mL of methanol/deionized water (9 : 1) and centrifuged at 12000 rpm for 15 min. e last method used different kinds of organic solvents (methanol or acetonitrile) to precipitate protein. e plasma sample (0.1 mL) was added in a threefold organic solvent and vortexed at 2000 rpm for 1 min. en, the sample was centrifuged at 12000 rpm for 15 min to obtain the supernatant, which was transferred to a clean tube and dried in an N 2 stream of nitrogen at room temperature. e residues were reconstituted and centrifuged under the sample condition mentioned above. All the final supernatant was injected into the UHPLC-Q-Exactive Orbitrap for data acquiring.
Each organ, including the heart, liver, spleen, lung, kidney, and brain, was cut into pieces and 0.2 g of mixed samples of each organ was homogenized in 5 volumes of icecold saline and centrifuged at 14,000 rpm for 10 min to get the supernatant as tissue samples. A total of 1 mL tissue sample was further processed by the second method described above.

Instruments and Conditions.
e chromatographic analysis of all samples was performed with a ermo Scientific Hypersil GOLD C18 column (100 × 2.1 mm, 1.9 µm) using an Ultimate 3000 UPLC system ( ermo Fisher Scientific, San Jose, CA, USA). 0.1% formic acid aqueous solution (solvent A) and acetonitrile (solvent B) were used as mobile phases with a flow rate of 0.30 mL/min. e flow rate was set at a linear gradient as follows: 0-2 min, 2% B; 2-3 min, 2-25% B; 3-8 min, 25-30% B; 8-12 min, 30-60% B; 12-15 min, 60-80% B; 15-17 min, 80-2% B; 17-20 min, 2% B. e injection volume was 2 μL. e high-resolution, accurate-mass analysis was performed on Q-Exactive Focus Orbitrap MS ( ermo Electron, Bremen, Germany) with heated electrospray ionization (ESI) source in the negative ions mode. e optimized tune method was set as follows: the flow rate of sheath gas (nitrogen, purity ≥ 99.99%) and auxiliary gas (nitrogen, purity ≥ 99.99%) was set at 30 and 10 arbitrary units, respectively; the temperatures of auxiliary gas heater and capillary were 350 and 320°C, respectively; the voltage of spray was 35 KV; and the S-lens RF level was set at 50. e full MS scan data were acquired at a mass range of m/z from 100 to 1000 at a resolving power of 70,000 to screen potential metabolites. e MS 2 data were obtained at parallel reaction monitoring scanning mode for the identification of metabolites. e collision energy of collision gas (nitrogen, purity ≥ 99.999%) for collision-induced dissociation (CID) was adjusted to 30%.

Data
Processing. All raw data were processed by the ermo Xcalibur software version 4 and the Compound Discover software version 3. e chemical formulas for all parent and fragment ions of the selected peaks were speculated by the accurate mass using a formula predictor by setting the parameters as follows: 3], and N[0-3]. e maximum mass tolerance was set at 5 ppm. Blank biological samples were used as controls for comparison with the analytic samples, and they were all processed under the same conditions.

Analytical Strategy.
In this study, all the plasma samples were prepared by the three methods mentioned above to obtain the supernatant, which was injected into UHPLC-Q-Exactive Orbitrap MS to acquire the high-resolution full mass data with the full mass scanning mode. en, data mining was processed by the Compound Discover workstation using the metabolism workflow template to detect the potential ions based on the biotransformation reactions. Subsequently, the MS 2 of potential ions was acquired based on the parallel reaction monitoring mode triggered by the potential ions. Finally, the AO metabolites were characterized based on the retention time, accurate full mass, the fragmentation of MS 2 , and bibliography.

Comparison of the Different Sample Preparation Methods.
In this study, three methods were employed to prepare the samples, and then all of them were applied to the UHPLC-Q-Exactive Orbitrap MS under the same condition. According to the retention time, accurate full mass, the fragmentation of MS 2 , and bibliography, a total of 21, 36, and 36 metabolites were screened and detected in the rat plasma using methods 1, 2, and 3, respectively, as shown in Table 1S.
To the best of our knowledge, the above-mentioned methods have been widely used to pretreat a biological sample, holding a great significance in the sample pretreatment. On the basis of the results, the second and the last methods displayed the same results of sample pretreatment, while sample pretreatment by method 1 showed fewer metabolites. After comparison, the second method was chosen as the final preparation method due to the economic solvent.

Identification of AO Metabolites.
A total of 36 metabolites (AO included) were tentatively characterized by means of the UHPLC-Q-Exactive Orbitrap MS with the PRM mode. Among them, 22 metabolites were detected for the first time.
e high-resolution extraction ion chromatography of these metabolites is shown in Figure 2. e detailed information of these metabolites, including the retention time, the accurate mass, and fragmentation ions, is listed in Table 1.
Metabolite 1 was accurately identified as AO by comparing the retention time, accurate mass, and MS 2 data with the reference substance. Metabolites 2 and 3 possessed the same MS information, including MS 1 and MS 2 and different chromatography behavior with the AO; thus, they were identified as isomers of AO. Metabolites        , respectively. erefore, they were tentatively characterized by glucuronidation and demethylation of AO. Likewise, metabolites 32-33 were plausibly characterized by diglucuronidation and demethylation of AO.

Distribution of AO Metabolites in Rats' Tissues.
To the best of our knowledge, the distribution of AO metabolites was investigated for the first time. A total of 16, 15, 10, 15, 16, and 9 metabolites were detected and identified in the heart, liver, spleen, lung, kidney, and heart, respectively (Table 1S). Most of the metabolic reactions, including reactions of Phases I and II, were observed in these organs, which indicated that AO metabolites are widely distributed in all these organs. Metabolites 1,5,11,12,20,21,28, and 36 were distributed in all these organs, suggesting that these metabolites might be the effective form of AO metabolites for exerting pharmacological effects.

Conclusion
In this study, an effective strategy based on UHPLC-Q-Exactive Orbitrap MS combined with PRM data acquiring was established for the detection and identification of AO metabolites in rats. Finally, a total of 36 metabolites, including phase I and phase II, were characterized in the rat plasma, out of which 22 were reported for the first time. e corresponding reactions, including demethoxylation, hydroxylation, demethylation, sulfation, glucuronidation, and combination reactions, were observed in this study. e study demonstrated that this strategy is useful for the detection of AO metabolites in various biological samples.

Data Availability
e data used to support the finding of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.

Authors' Contributions
Shihan Qin and Yuan Xu contributed equally to this work.