Detection and characterization of the metabolites of rutaecarpine in rats based on ultra-high-performance liquid chromatography with linear ion trap-Orbitrap mass spectrometer

Abstract Context: Rutaecarpine is an active indoloquinazoline alkaloid ingredient originating from Evodia rutaecarpa (Wu-zhu-yu in Chinese), which possesses a variety of effects. However, its metabolism has not been investigated thoroughly yet. Objective: This study develops a highly sensitive and effective method for detection and characterization of the metabolites of rutaecarpine in Sprague–Dawley (SD) rats. Materials and methods: In this study, an efficient method was developed using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer (UHPLC–LTQ-Orbitrap MS) to detect the metabolism profile of rutaecarpine in rat plasma. First, a blood sample (1 mL) was withdrawn 2 h after oral administration of rutaecarpine in SD rats (50 mg/kg). Second, the blood was centrifuged at 4000 rpm for 10 min and pretreated by solid-phase extraction method. Third, 2 μL of the plasma was injected into UHPLC–LTQ-Orbitrap MS for analysis. Finally, the metabolites of rutaecarpine were tentatively identified based on accurate mass measurements, fragmentation patterns and chromatographic retention times. Results: A total of 16 metabolites (four new metabolites, viz., dihydroxylation and sulphate conjugation products of rutaecarpine (M8–M11)) as well as parent drug itself, including three phase I and 12 phase II metabolites were detected and identified in rat plasma. Hydroxylation, sulphate conjugation and glucuronidation were confirmed as the primary metabolic pathways for rutaecarpine in rat plasma. Discussion and conclusion: These results provide an insight into the metabolism of rutaecarpine and also can give strong indications on the effective forms of rutaecarpine in vivo.


Introduction
Rutaecarpine is an active indoloquinazoline alkaloid ingredient originating from Evodia rutaecarpa (Wu-zhu-yu in Chinese). It has been used extensively as traditional Chinese medicine (TCM) for decades and is officially listed in the Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission 2015). In recent years, despite a lack of knowledge in mechanistic details, pharmacological studies have demonstrated that it possesses antithrombotic, antianoxic, hypotensive, anti-inflammatory, uterotonic, thermoregulatory, anti-obesity and vasodilatory effects (Liu & Ho 1999;Kobayashi et al. 2001;Liao et al. 2011;Yu et al. 2013).
Previous pharmacokinetic studies of rutaecarpine indicated that it had a low oral bioavailability, which was attributed to the first-pass metabolism or poor absorption from the gastrointestinal tract (Li 2005). Currently, due to the limitation of analytical techniques, the metabolites of rutaecarpine have not been fully investigated (Ueng et al. 2005(Ueng et al. , 2006Jan et al. 2006). For instance, only 16 metabolites in urine and 11 in feces were detected and identified Kim et al. 2008), whereas no metabolite was characterized in the plasma. Therefore, it is important to detect and identify its metabolism profile of rutaecarpine in plasma, which can help to further understand the mechanism of action of rutaecarpine.
During the past decade, liquid chromatography/mass spectrometry was the main analytical method for the structural characterization of drug metabolites in vivo and in vitro (Lin et al. 2012;Szultka et al. 2014). Ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometer (UHPLC-HRMS) such as UHPLC coupled with linear ion trap-Orbitrap mass spectrometer (LTQ-Orbitrap MS) significantly contributed to the characterization of drug metabolites due to its higher separation and resolution capacity in a shorter time (Du et al. 2011;Cai et al. 2015).
In the present study, a total of 16 metabolites (four new) as well as the parent drug itself, including three phase I and 12 phase II metabolites in rat plasma were detected and identified based on accurate mass measurements, fragmentation patterns and chromatographic retention times.

Chemicals and reagents
Rutaecarpine (Figure 1), with a purity of >98% by HPLC analysis was isolated from a 95% ethanol extract of Euodiae fructus (identified by professor Wei-feng Dong, Department of Pharmacy, Hunan University of Medicine) in our laboratory. Its structure was identified by comparing UV, MS and NMR data with what is known in the literature. The structure is shown in Figure 1. Grace Pure TM SPE C18 phase extraction cartridges (200 mg/3 mL, 59 lm, 70 Å) were purchased from Grace Davison Discovery Science TM (Deerfield, IL), and all the water used in experiments was purified by a Milli-Q water purification system (Millipore, Billerica, MA). Acetonitrile of HPLC-grade was purchased from Fisher (Fisher, NJ). All other chemicals and regents were of analytical grade and commercially available without further purification.

Animals and drug administration
Male Sprague-Dawley (SD) rats (250 ± 20 g, Beijing Weitong Lihua Experimental Animals Company, Beijing, China) were acclimatized in controlled environmental conditions (temperature, 24 ± 2 C; relative humidity, 70 ± 5%) for a week and were allowed free access to food as well as water ad libitum. Rats were randomly divided into two groups after a fast of more than 12 h: Group A (n ¼ 3); drug group for drug plasma and Group B (n ¼ 3); control group for blank plasma. All animal experiments were performed in accordance with the approved animal protocols and guidelines established by medicine ethics review committee for animal experiments of Hunan University of Medicine. Rutaecarpine was suspended in carboxymethylcellulose sodium (CMC-Na) aqueous solution and orally administered to group A at a dose of 50 mg/kg body weight, and an equivalent 0.5% CMC-Na solution was administered by orally gavage to group B. Blood samples (2 mL) were withdrawn in heparinized centrifuge tubes at 2 h following oral administration and centrifuged at 4000 rpm for 10 min to obtain plasma. All plasma samples were stored at À20 C until further pretreatment and analysis.

Sample preparations
Plasma samples were pretreated by solid-phase extraction method before UHPLC-MS analysis. An SPE cartridge was pretreated with 5 mL of water, 5 mL of methanol and 5 mL of water, successively. A sample of plasma (1 mL) was processed on a pre-activated solid phase extraction C18 column, and then eluted with 5 mL of water followed by 5 mL of methanol. The methanol eluent was collected and dried under nitrogen gas at room temperature. The residue was re-dissolved in 100 lL of methanol and 2 lL supernatant after centrifugation (12,000 rpm, 30 min at 4 C) was injected into UHPLC-ESI-LTQ-Orbitrap MS for analysis.
The ESI mass spectra (MS) were acquired in a negative mode by full scan. The MS analyses were performed under optimized conditions, using a spray voltage of 4.0 kV, a capillary voltage of 25 V, a tube lens voltage of 110 V, a curved desolvation line and heat block temperature, an aux gas (nitrogen) flow rate of 5 arb, a sheath gas (nitrogen) flow rate of 30 arb and capillary temperature of 350 C. The spectra were recorded in the range of m/z 100-800 for MS resolution of the Orbitrap mass analyser at 30,000 units. Data-dependent MS/MS scanning was performed to minimize total analytical time as it can trigger fragmentation spectra of target ions. The collision energy for collision induced dissociation (CID) was adjusted to 30% of maximum, and the isolation width of precursor ions was m/z 2.0 Da.

Fragmentation pathway of rutaecarpine
In order to facilitate the structural identification of metabolites, the MS n fragmentation pattern of rutaecarpine was investigated in the negative mode detection by ESI (Gao et al. 2012). The parent ion showed a deprotonated ion [M À H] À at m/z 286.0982 (2.4 ppm, C 18 H 12 ON 3 ). Fragmentation of this parent ion provided a characteristic fragment ion at m/z 169.0764 (2.2 ppm, C 11 H 9 N 2 ) by the loss of the moiety (C 7 H 3 ON). The fragment ion at m/z 142 was produced by the loss of C 7 H 3 ON þ CNH from the parent ion, which is useful information in metabolite identification. Besides, the fragment at m/z 142 can be formed by the loss of CNH from the ion at m/z 169 in the MS 3 spectra. The proposed fragmentation pattern of rutaecarpine is illustrated in Figure 2.

Detection and structural elucidation of metabolites
After comparison of the high-resolution EIC (HREIC) of drug samples with corresponding control samples, a total of 16 metabolites as well as the parent drug itself were detected and identified. The HREICs of drug samples are shown in Figure 3. The chromatographic and mass spectrometric data of the parent drug and its metabolites are shown in Table 1.

Metabolite M0
Metabolite M0 was unambiguously identified as rutaecarpine by comparing the retention time and MS with the authentic reference.

Metabolites M1, M2, M4, M5, M6 and M7
Metabolites M1 and M2 were eluted at 17.64 and 18.78 min with the quasi-molecular ions of m/z 302.0932 (2.6 ppm, C 18 H 12 O 2 N 3 ) and m/z 302.0933 (3.0 ppm, C 18 H 12 O 2 N 3 ), respectively; this was 16 Da more than that of parent drug, suggesting that they were hydroxylated products of rutaecarpine. By comparing the data in the literature , they were presumed to be 10-hydroxyrutaecarpine and 3-hydroxyrutaecarpine, respectively. Metabolites M4-M7, possessing deprotonated molecular ions [M-H]at m/z 382.0501 (2.4 ppm, C 18 H 12 O 5 N 3 S), m/z 382.0505 (3.3 ppm, C 18 H 12 O 5 N 3 S), m/z 382.0505 (3.3 ppm, C 18 H 12 O 5 N 3 S) and m/z 382.0505 (3.3 ppm, C 18 H 12 O 5 N 3 S), were detected at 17.02, 18.10, 20.04 and 22.95 min, respectively. The fragment ion at m/z 302 was produced by the loss of sulphate group (80 Da) from the precursor ion at m/z 382. Based on this analysis, they were plausibly deduced as hydroxylation and sulphate conjugation products of rutaecarpine .   , they were presumed to be hydroxylation and glucuronide products of rutaecarpine.

Proposed metabolic pathways of rutaecarpine
In this study, 16 metabolites (four new) as well as the parent drug were detected in the plasma. The proposed major metabolic pathways of rutaecarpine in the rat plasma are shown in Figure 4. In general, the metabolism of rutaecarpine in vivo undergoes hydroxylation metabolic reactions (M1-M2) first, followed by dihydroxylation (M3), and then hydroxylation þ sulfate conjugation (M4-M7), dihydroxylation þ sulphate conjugation (M8-M11) and finally hydroxylation þ glucuronide conjugation (M12) that took place on the position of hydroxyl groups.

Conclusion
The metabolic profile of rutaecarpine in plasma was investigated following oral administration of a single dose of rutaecarpine to SD rats using a UHPLC-LTQ-Orbitrap MS for analyses. By online LC-MS n data acquisition and offline data processing methods of the software xcalibur 2.1, a total of 16 metabolites (four new) as well as the parent drug itself, including three phase I and 12 phase II metabolites were detected and identified based on accurate mass measurements, fragmentation patterns and chromatographic retention times. The metabolic reactions of rutaecarpine in rats were hydroxylation, dihydroxylation, hydroxylation þ sulphate conjugation, dihydroxylation þ sulphate conjugation, and hydroxylation þ glucuronidation. In conclusion, profiling the metabolites of rutaecarpine in rats could in future provide comprehensive insights on its pharmacological effects, metabolic fate in vivo and effective forms.

Disclosure statement
The authors have declared no conflict of interest.