Identification of In Vivo Metabolites of Dictamnine in Mice Using HPLC-LTQ-Orbitrap Mass Spectrometry

Dictamnine (4-methoxyfuro[2,3-b]quinolone, DIC), a common furoquinoline alkaloid in the family of Rutaceae, showed diverse biological activities. To investigate the in vivo metabolic pathways of DIC, metabolism of DIC in mice was studied using a high-performance liquid chromatography coupled to electrospray ionization of hybrid linear trap quadrupole orbitrap (HPLC-LTQ-Orbitrap) mass spectrometer. Nine metabolites were identified in the DIC-treated mouse urine, plasma, and fecal samples, of which two were identified as new metabolites. The major metabolic pathways of DIC in animal and human liver microsomes were confirmed in the present study, including o-demethylation, monohydroxylation, N-oxidation, and 2,3-olefinic epoxidation pathways. For the first time, a mono-acetylcysteine conjugate of DIC (M9) was detected from DIC-treated mouse urine and plasma samples, and 4-methoxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (M10) and 2-(2,8-dihydroxy-4-methoxyquinolin-3-yl)acetaldehyde (M11) were identified as new metabolites of DIC; furthermore, using an in vitro human fecal incubation model, furo[2,3-b]quinolin-4-ol (M1) was verified to be a microbial demethylated metabolite of DIC. Collectively, the present study provided new information on the in vivo metabolic fate of DIC.

Our previous studies have demonstrated that DIC is a promising drug candidate with favorable oral pharmacokinetic profiles [12,13]. Very recently, using in vitro microsomal incubation systems, we systematically studied the metabolites of DIC in human and animal liver microsomal incubation systems [14], and we found that the metabolic fate of DIC in human liver microsomes was about the same as that in mouse liver microsomes, while with some differences compared with that in rat, dog, and monkey liver microsomes. We elucidated that the o-demethylation, monohydroxylation, N-oxidation, and 2,3-olefinic epoxidation pathways are the major metabolic routes of DIC [14]. In a separate study [15], a N-acetylcysteine (NAC) conjugate which was derived from the reactive DIC metabolite, 2,3-epoxide DIC, was detected in the rat liver microsomal incubation system, as well as in DIC-treated rat urine samples.
It is also remarkable to note that there are no reports about the in vivo metabolism study of DIC in either humans or mice. Choosing the most relevant animal species to conduct studies is essential for researchers to extrapolate results from studies performed in animals onto human [16][17][18][19]. In the preset study, based on the previous results [14,15], the in vivo metabolism study of DIC was performed on mice. A powerful hybrid linear quadrupole ion trap orbitrap (LTQ-Orbitrap) mass spectrometer equipped with a high-performance liquid chromatography (HPLC) system was used for the structural elucidation of the metabolites.

Materials and Methods
2.1. Reagents. DIC, purity higher than 98%, was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). HPLC-MS-graded acetonitrile (ACN), methanol (MeOH), and formic acid were obtained from Fisher Scientific (Pittsburgh, PA, USA). Purified water was obtained from a Milli-Q system (Millipore, Bedford, USA).

Treatment of Mice and Sample Collections.
Experiments with mice were carried out with male Kunming mice (25 ± 2 g) which were obtained from the Experimental Animal Center of Qingdao University (Qingdao, China). All mice were housed 5 per cage with the 12 h light/dark cycle at an ambient temperature of 20 ± 2°C with a relative humidity of 50 ± 10%. Mice were allowed to acclimate for at least 1 week prior to the start of the experiment. Mice were fed with AIN-93 semipurified diet and were allowed ad libitum food and water throughout the study. Metabolic cages were used for the collection of twenty-four-hour mouse urine and fecal samples. In brief, DIC in DMSO or DMSO only were gavaged to mice individually, and mouse feces and urine were collected using metabolic cages (5 mice per cage) for 24 h after administration of DIC (the treated group, n � 5, 5 mg/kg body weight) or vehicle (the control group, n � 5).
e mouse blood sample was collected from anesthetized mice by a cardiac puncture at 2 h after administration of vehicle or DIC, and the whole blood sample was centrifugated at 8,000 × g for 10 minutes at 4°C to isolate plasma. Before analysis, all samples were stored at −80°C.

Fecal, Urinary, and Plasmatic Sample Preparation.
For acquisition of the metabolic profiles, six pieces of each fecal sample (control and treated) were chosen and homogenized with 1 mL of MeOH/H 2 O/acetic acid (90/10/0.1, v/v/v) solution for 3 minutes and then centrifuged at 12,000 × g for 10 minutes at 4°C. An aliquot (200 μL) of the supernatant was picked up and diluted for 20 times before analysis. e urine and plasma samples were deconjugated by the enzyme as described previously with slight modifications [20,21]. In brief, 100 μL from each sample (control and treated) was incubated with β-glucuronidase (250 U) and sulfatase (3 U) for 90 min in a water bath at 37°C. e reaction was quenched by adding an equal volume of MeOH containing 1% acetic acid and centrifuged at 12,000 × g for 10 minutes at 4°C. Aliquots of the supernatant were stored at −20°C before analysis.

In Vitro Fecal Batch Fermentation Experiments.
ree healthy volunteers (mean age, 32.5-years-old; mean weight, 69.5 kg; nonsmokers) were involved in this study, all of them were antibiotic-free for over 6 months before the study begins, and they had not taken polyphenol-rich foods for over two days before the stool sample collection. Stool samples were collected and transported immediately to the anaerobic condition and were processed within 2 h. Under anaerobic conditions, 50 g of the fresh collected stool sample was homogenized in a sterilized stomacher bag with 100 mL of water containing 0.05% peptone. e particulate materials were removed by centrifugation at 3000 rpm for about 2.5 min, and then the supernatant of the stool slurry was mixed with 35% presterile glycerol and stored at −80°C before use. For the in vitro fermentation experiment, a pooled human stool slurry was made by direct mixing of an equal volume of the stool slurry from each subject. e fermentation basal medium contains about 4% of Tween 80, and 2% of peptone was freshly prepared and autoclaved before study. Five μL of DIC solution (5 mg/mL in DMSO), 5 mL of fermentation medium, and 0.5 mL of pooled human stool slurry were mixed and then aliquoted into 3 sets of samples (∼1.5 mL each) to represent 0, 12, and 24 h time points. Under anaerobic conditions, samples were fermented at 37°C and harvested at the corresponding time intervals. Once harvested, 450 μL of the supernatant was picked up and quenched by adding 900 μL of acetonitrile (0.2% acetic acid). Samples were centrifuged at 12,000 × g for 10 min, and then aliquots (200 μL) of the supernatants were transferred to HPLC vials for analysis.

HPLC-LTQ-Orbitrap Conditions.
HPLC was performed on a HPLC system comprised degassers, a quaternary pump, a column compartment, and an autosampler from ermo-Finnigan ( ermo Electron Co., USA). A Phenomenex Gemini C18 column (3.0 mm × 150 mm; 5 μm; Torrance, CA, USA) was used for separation with column temperature set at 35°C. e HPLC conditions were the same as we described before [14]. In brief, the composition of the mobile phase was 5% water with 95% methanol containing 0.1% formic acid as mobile phase A and 95% water with 5% methanol containing 0.1% formic acid as mobile phase B. Gradient elution program was initiated with 10% B for 5 min, then increased to 25% from 5 to 10 min, to 60% from 10 to 30 min, to 100% from 30 to 40 min, and then held constant for 5 min. e column was equilibrated with 10% B for 5 min between each run. e flow rate was set at 0.3 mL/ min, and 10 µL was injected for each sample.
A Finnigan LTQ-Orbitrap XL mass spectrometer coupled with an electrospray ionization (ESI) interface ( ermo Electron, Bremen, Germany) was used for the mass spectra collection. Nitrogen was used as a sheath and auxiliary gas with 30 and 10 arbitrary units, respectively, and helium was applied as the collision gas. e tube lens and capillary voltage were set at 65 V and 250 V, respectively. e mass spectrometer was set to repeat a cycle consisting of a full MS scan followed by an MS/MS scan. Mass spectra were collected from m/z 50 to 500 amu in the positive ionization mode. Collision-induced dissociation (CID) energy was set at 35 for MS n analysis with an isolation width set at 1.5 amu and activation time set at 30 ms, respectively. Data acquisition and analysis were performed using Xcalibur software (2.2 version, ermo Electron, San Jose, CA, USA).

Metabolic Fate of DIC in Mice.
In present study, HPLC-LTQ-Obitrap/MS was used to explore the major metabolites of DIC in samples. Extracted ion chromatograms (EIC) of the metabolites detected from the DIC-treated mouse fecal, plasmatic, and urinary samples are shown in Figure 1. Compared to the samples collected from vehicle-treated mice, nine major metabolites were observed. M1-M5 and M8 were numbered according to our previous study, and new identified metabolites (M9-M11) were numbered sequentially to their chromatographic retention times. By comparing the LC/MS n data and retention times to our previously microsomal studies and other literatures [14,22,23], six metabolites were unequivocally identified as furo [  Journal of Analytical Methods in Chemistry those of DIC, indicating that the structure of DIC remains unchanged [14,15]. is result suggests the NAC was conjugated to the furan ring.

Metabolism of DIC by Human Fecal Gut Microbiota.
To study the impact of gut microbiota for the in vivo metabolism of DIC, DIC was incubated with the pooled human stool slurries obtained from three healthy individuals. Samples were harvested as a function of time and analyzed by LC-MS for the elucidation of the microbialderived metabolites. Figure 5 shows a representative extracted ion chromatograms of DIC metabolites after incubating with the pooled human stool slurry. DIC was digested progressively with increasing time. Compared with the control sample (collected from 0 h time point), a new peak, retention time at 20.6 min, was detected from the sample collected at the time point of 12 h, and the peak area of this peak increased with increasing time. is peak was identified as furo [

Concluding Remarks
In summary, our present study showed that DIC, a common furoquinoline alkaloid belongs to the family of Rutaceae, can be extensively metabolized in mice. In total, nine metabolites were identified in mouse urinary, plasmatic, and fecal samples, and two of those (M10 and M11) were identified for the first time. e metabolic profile of DIC in the mouse was finally elucidated ( Figure 6).
Additionally, our present study showed that the gut microbiota intermediates the o-demethylation of DIC, which results in the demethylation metabolite of DIC, M1. Finally, the metabolic pathway of DIC in mice was proposed, and these data are significant for further researches of DIC and allows intelligent assessment of preclinical toxicology and/or in vivo bioactivity studies performed on mice.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.   Journal of Analytical Methods in Chemistry