Profiling and Pharmacokinetic Studies of Alkaloids in Rats After Oral Administration of Zanthoxylum nitidum Decoction by UPLC-Q-TOF-MS/MS and HPLC-MS/MS

Zanthoxylum nitidum (Roxb.) DC (Rutaceae), called as “liangmianzhen” in China, is well known for its anti-inflammation and analgesic effect. Alkaloids are its main active constituents. However, little has been known about the absorption of main alkaloids in vivo. In this study, an ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry was employed for identification of absorbed alkaloids in rats after oral administration of Z. nitidum decoction. By analyzing the fragmentation patterns, a total of nineteen alkaloids were exactly or tentatively identified in rat plasma after treatment, of which magnoflorine, α-allocryptopine, and skimmianine are dominant. Moreover, a high performance liquid chromatography coupled mass spectrometry method was developed for simultaneous quantification of magnoflorine, α-allocryptopine, and skimmianine, and successfully applied to pharmacokinetic study in rats after oral administration of Z. nitidum decoction. The research would contribute to comprehensive understanding of the material basis and function mechanism of Z. nitidum decoction.


Identification of Absorbed Alkaloids of Z. nitidum Decocotion in Rat Plasma
To identify the absorbed alkaloids in vivo, the rat plasma after oral administration of Z. nitidum decotion was analyzed using the target (Table S1) and untarget strategy reported by Zhang et al. [26]. As a result, a total of 19 prototype alkaloids were identified, including 2 aporphinoid, 3 protopine, 7 benzophenanthrindine, and 7 quinoline alkaloids (Table 1). Among 19 compounds, magnoflorine, α-allocryptopine, nitdine, chelerythrine, and skimmianine were unambiguously characterized by comparison with authentic standards. Other compounds were tentatively deduced based on accurate mass of quasimolecular, MS 2 spectra and fragmentation pathway, and some isomers were further differentiated by considering relative retention time and molecular polarity. The total ion chromatograms (TICs) of these components are shown in Figure 1 and their chemical structures are shown in Figure 2. The extract ion chromatograms (EICs) and MS 2 spectra are given in Figure S1.   To facilitate alkaloid identification, 5 authentic standards representing 5 known alkaloids in Z. nitidum, including one aporphine (magnoflorine), one protopine (α-allocryptopine), two benzophenanthrindine (nitidine and chelerythrine), and one quinoine (skimmianine) were selected and analyzed thoroughly to illustrate the proposed fragmentation pathways for references . Among them, the fragment pathway of skimmianine is given in our previous published reference [27].
Magnoflorine was eluted at 4.2 min with the parent ion at m/z 342.1706 (C20H24NO4 + ). The fragment ion at m/z 297.1121 (C18H17O4 + ) was attributed to the elimination of (CH3)2NH, which might be an important characteristic of aporphine alkaloid fragmentation pathway [28]. Subsequently, the fragment ion at m/z 265.0859 (C17H13O3 + ) was observed as the base peak due to the loss of CH3OH. Because of the electron-withdraw inductive effect and the minimal energy of ion, the expulsion of CH3OH could occur from vicinal hydroxyl and methoxy groups on C1 and C2 [29]. The fragment ion at m/z 237.0910 (C16H13O2 + ) was produced by the neutral loss of CO from the fragment ion at m/z  To facilitate alkaloid identification, 5 authentic standards representing 5 known alkaloids in Z. nitidum, including one aporphine (magnoflorine), one protopine (α-allocryptopine), two benzophenanthrindine (nitidine and chelerythrine), and one quinoine (skimmianine) were selected and analyzed thoroughly to illustrate the proposed fragmentation pathways for references . Among them, the fragment pathway of skimmianine is given in our previous published reference [27].
Magnoflorine was eluted at 4.2 min with the parent ion at m/z 342.1706 (C20H24NO4 + ). The fragment ion at m/z 297.1121 (C18H17O4 + ) was attributed to the elimination of (CH3)2NH, which might be an important characteristic of aporphine alkaloid fragmentation pathway [28]. Subsequently, the fragment ion at m/z 265.0859 (C17H13O3 + ) was observed as the base peak due to the loss of CH3OH. Because of the electron-withdraw inductive effect and the minimal energy of ion, the expulsion of CH3OH could occur from vicinal hydroxyl and methoxy groups on C1 and C2 [29]. The fragment ion at m/z 237.0910 (C16H13O2 + ) was produced by the neutral loss of CO from the fragment ion at m/z Chemical structure of the alkaloids in rat plasma after oral administration of Z. nitidum decoction.
Because of the electron-withdraw inductive effect and the minimal energy of ion, the expulsion of CH 3 OH could occur from vicinal hydroxyl and methoxy groups on C1 and C2 [29]. The fragment ion at m/z 237.0910 (C 16 [17]. Compounds 16 and 18 were deduced to be isoarnottianamide and arnottianamide [5]. These two compounds were derivatives of benzophanthridine containing N-methylformamide group, whose mass spectrum a minor peak corresponding to the loss of HCONHCH 3  Besides skimmianine, six quinoline alkaloids (5, 6, 9, 12, 14, 15) were detected. Compounds 5, 6, and 9 were reported for the first time in Z. nitidum.

Quantitative Method Validation
After oral administration of Z. nitidum decoction, a total of 19 prototype alkaloids were identified. Considering the quality control component of Z. nitidum suggested by Chinese Pharmacopoeia 2015, content in Z. nitidum, plasma exposure level and the availability of reference standard, magnoflorine, α-allocryptopine, nitdine, chelerythrine, and skimmianine were selected to perform pharmacokinetic experiments by HPLC-MS/MS. However, the results of the preliminary experiment showed that nitidine and chelerythrine had poor absorption, as reported in the literatures [34,35]. Moreover, obvious interference was observed from endogenous material at the rentention times of nitidine and chelerythrine during the chromatographic separation, despite various sample preparation methods were applied. Finally, magnoflorine, α-allocryptopine, and skimmianine were selected for the further pharmacokinetic study. The corresponding quantification method using HPLC-MS/MS were developed.
The typical chromatograms of blank plasma, blank plasma spiked with three analytes and internal standard (IS), and plasma after oral administration of Z. nitidum decoction were shown in Figure 6. No obvious interference was observed from endogenous material at the rentention times of analytes and IS. As shown in Table 2, magnoflorine, α-allocryptopine, and skimmianine showed good linearity (r > 0.999) over the linear range. The lower limit of quantification (LLOQ) of magnoflorine, α-allocryptopine and skimmianine were 2, 2, and 0.5 ng/mL, respectively. The intra-and inter-day precision and accuracy were summarized in Table 3. All analytes displayed relative standard deviation (RSD%) below 11.23% and relative error (RE%) ranged from 8.05% to 11.23%, which were within the acceptable criteria. The extraction recovery of magnoflorine, α-allocryptopine and skimmianine were in the range of 89.87-98.32% and IS was 93.90%. The matrix effects of three analytes were in the range of 92.73-108.46% and IS was 93.88%. The stability of analytes under four storage conditions were assessed and the results were listed in Table 4. All analytes exhibited RSD% below 11.32% and RE% ranged from 10.00% to 12.53%, indicating that the analytes were stable. In conclusion, the developed method was validated and satisfactory for pharmacokinetic study.    Table 3. Precision, accuracy, extraction recovery and matrix effect of analytes in rat plasma (n = 6).

Pharmacokinetic Study
The validated HPLC-MS/MS method was successfully applied for the pharmacokinetic study of magnoflorine, α-allocryptopine, and skimmianine in rat plasma after oral administration of Z. nitidum decoction. The plasma concentration-time curve was shown in Figure 7. The main pharmacokinetic parameters were processed by Drug and Statistics (DAS) 2.0 software and listed in Table 5.

Pharmacokinetic Study
The validated HPLC-MS/MS method was successfully applied for the pharmacokinetic study of magnoflorine, α-allocryptopine, and skimmianine in rat plasma after oral administration of Z. nitidum decoction. The plasma concentration-time curve was shown in Figure 7. The main pharmacokinetic parameters were processed by Drug and Statistics (DAS) 2.0 software and listed in Table 5.  As shown in Figure 7, the three components were absorbed rapidly after oral administration, with Tmax ranged from 0.38 to 1.05 h. The Cmax of magnoflorine and α-allocryptopine were about ten  As shown in Figure 7, the three components were absorbed rapidly after oral administration, with T max ranged from 0.38 to 1.05 h. The C max of magnoflorine and α-allocryptopine were about ten times higher than that of skimmianine. Combined with their contents in Z. nitidum decoction (6.7, 1.1 and 0.4 mg/mL), the absorption rate of α-allocryptopine might be the highest of these three alkaloids. The T 1/2 of magnoflorine, α-allocryptopine and skimmianine were 3.24 ± 1.31, 0.78 ± 0.17 and 5.99 ± 1.62 h, respectively. The total exposure area under curve (AUC) 0-∞ of magnoflorine was the largest of the three components. The relatively higher plasma concentration and AUC 0-∞ indicated that magnoflorine and α-allocryptopine might have favorable drug-like properities.
Magnoflorine has been reported for its diverse pharmacological properties, such as anti-inflammatory, anti-bacteria and immunomodulatory effects [36][37][38][39]. Some of these properties might contributed to anti-inflammation and analgesic effect of Z. nitidum decoction in clinic use. Limited information is known about pharmacological properties of α-allocryptopine. Available studies indicate that α-allocryptopine possesses antiarrhythmic effects [40][41][42]. Skimmianine has been reported to possess anti-inflammatory and non-narcotic analgesic effects etc. [43][44][45], but its plasma concentration and AUC 0-∞ was relatively lower in this study. Whether there exist synergistic action of them needs further investigation.
As for nitidine, due to poor absorption, its functional mechanism in vivo needs further study.

Chemical, Reagents and Materials
The roots of Z. nitidum were collected from Guangdong, China, and authenticated by RT Zhan. Magnoflorine
MS spectra were achieved on an AB SCIEX Triple TOF 5600 (AB Sciex Pte. Ltd., Singapore, Singapore) with electrospray ionization (ESI) source in positive mode. The following parameters of mass spectra were used: source temperature at 500 • C; nebulizer and heater gas pressure at 50 psi; curtain gas pressure at 40 psi; ion spray voltage at 5500 V; declustering potential at 100 V, collision energy 10 eV, and mass range 100-800 amu. The collision energy in "product ion" scan was set at 35 v with a collision energy spread of 10 eV. Data acquisition was controlled with AB SCIEX Analyst TF (Version 1.7) software (AB Sciex Pte. Ltd., Singapore, Singapore). Data processing was performed with Peakview (Version 2.0) software (AB Sciex Pte. Ltd., Singapore, Singapore).
MS data were achieved on a triple-quadrupole mass spectrometer: TSQ Quantum Access (Thermo Fisher Scientific, Waltham, MA, USA) with electrospray ionization (ESI) source in positive mode. The instrument parameters were as follows: spray voltage at 3000 V, sheath gas and auxiliary gas with a flow of 30 and 5 arbitrary units, capillary temperature at 350 • C, collision gas pressure at 1.0 mTorr, skimmer offset at 2 V. The selected reaction monitoring (SRM) transitions used for MS analysis were as follows: m/z 342→297 for magnoflorine, 370→188 for α-allocryptopine, 260→227 for skimmianine, 356→192 for IS; the collision energies at 15 eV for magnoflorine, 30 eV for α-allocryptopine, skimmianine and IS. The data acquisition and processing was performed with Xcalibur 2.0 software (Thermo Fisher Scientific, Waltham, MA, USA).

Preparation of Samples
3.3.1. Preparation of Z. nitidum Decoction 100 g of the roots of Z. nitidum were weighed and decocted with 1.2 L of water for 3 h. The filtrate was collected and residue was decocted in 1.2 L of water for 2 h again. Subsequently, the filtrates from each decoction were combined and concentrated to 50 mL. The contents of magnoflorine, α-allocryptopine, nitidine, chelerythrine, and skimmianine in Z. nitidum decoction were 6.7, 1.1, 1.8, 3.5, and 0.4 mg/mL, respectively.

Qualitative Analysis
The 2 mL plasma sample was loaded on a pretreated SPE column which was eluted with 20 mL methanol followed by 20 mL water. After being washed off by 6 mL of water, the cartridge was eluted using 6 mL methanol. The methanol eluting was evaporated to dryness at 35 • C in vacuum using SpeedVac Concentration (Savant SPD 1010, Thermo scientific). The residue was reconstituted in 300 µL acetonitrile and water (50:50, v/v) and centrifuged at 13,000 rpm (15,493× g) for 15 min.
Quantitative Analysis 100 µL plasma sample, 10 µL IS solution (200 ng/mL) and 300 µL acetonitrile were added to a 1.5 mL eppendorf tube and vortex-mixed for 5 min, then centrifuged at 13,000 (15,493× g) rpm for 10 min. The supernatant was transferred into another eppendorf tube and evaporated to dryness at 35 • C in vacuum using SpeedVac Concentration ((RVC 2-18, Christ). The residue was reconstituted in 100 µL acetonitrile and water (50:50, v/v) and centrifuged at 13,000 rpm (15,493× g) for 15 min.

Calibration Samples and Quality Control Samples
The stock solutions of magnoflorine, α-allocryptopine and skimmianine were prepared in acetonitrile at 1.06, 1.02, and 0.98 mg/mL, respectively. A serious of working solutions were obtained by diluting with acetonitrile. The IS solution was prepared at a concentration of 200 ng/mL in acetonitrile.

Method Validation
Validation of the analytical method was assessed on selectivity, linearity, sensitivity, accuracy, precision, recovery, matrix effect, and stability according to Pharmacopoeia of the People's Republic of China 2015 guidelines.
The selectivity was assessed by analyzing the chromatograms of blank plasma of six different rats, a blank plasma with magnoflorine, α-allocryptopine, skimmianine and IS, and a plasma after dose. The linearity was determined by plotting the peak areas ratios (y) of each analyte to IS against the concentrations, and evaluated by least-squares linear regression. The lower limit of quantification (LLOQ) was defined as the lowest concentration point of the calibration curve (S/N > 10) with the accuracy within ±20% and precision lower than 20%. The accuracy and precision were evaluated by analyzing the six replicate QC samples on the same day (intra-day) and three consecutive days. The accuracy and precision were expressed as relative error (RE%) and relative standard deviation (RSD%), respectively. The extraction recovery was determined by comparing peak areas of the extracted QC samples with those of post-extracted spiked samples. The matrix effects was measured by calculated the analytes peak area ratios of post-extracted spiked samples to those of pure work solution. The stability was investigated by analyzing samples stability under diverse storage conditions: three freeze-thaw cycles, 8 h at 25 • C, −80 • C for 40 days and in autosampler for 24 h.

Animal Experiments
Male Sprague-Dawley rats (250 ± 20 g) used in this study were provided by the Experimental Animal Center of Guangzhou University of Chinese Medicine. The laboratory animal license number is SCXK 2013-0020. These animals were maintained in an air-conditioned animal facility at 23 ± 2 • C, with a humidity of 55% ± 5% and a 12 h light/dark cycle for 5 days before use. The rats had free access to water and a standard diet. Animal welfare and experimental procedures were strictly in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals in China and the related ethical regulations of Guangzhou University of Chinese Medicine.
For profile study, twelve rats were randomly divided into 2 group, blank control and experimental groups. Before administration, the rats were fasted for 12 h but allowed water ad libitum. Z. nitidum decoction was orally administered to experimental group at a dose of 15 mL.kg −1 (15 mL decoction equal to 30 g crude drug) body weight, while distilled water was orally administered to control group. The rats of experimental group were anesthetized at 0.5 h, 1 h, 2 h, 3 h, 4 h, and 6 h after dose, respectively. The blood samples were collected from aorta abdominalis in heparinized tube. All blood samples were then centrifuged at 3500 rpm (1274× g) for 15 min at 4 • C. Blank plasma samples were prepared following the same procedures. All samples were stored at −80 • C.
For pharmacokinetic study, six rats were orally administered Z. nitidum decoction at a dose of 5.4 mL kg −1 (5.4 mL decoction equal to 10.8 g crude drug) body weight. The blood samples were collected from orbital vein before dose and 0.17, 0.33, 0.67, 1, 2, 3, 4, 6, 8, 10, 12, and 24 after dose. The blood samples were then centrifuged at 3500 rpm (1274× g) for 15 min at 4 • C. The samples were stored at −80 • C. Data analysis was performed by Drug and Statistics (DAS) 2.0 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China).

Conclusions
In this study, the UPLC-Q-TOF-MS/MS method was used to identify the absorbed alkaloids in vivo after oral administration Z. nitidum decoction for the first time. The fragmentation pathway of magnoflorine, α-allocryptopine and nitidine was proposed, and a total of 19 alkaloids were exactly or tentatively identified in rat plasma after dose, including 2 aporphinoid, 3 protopine, 7 benzophenanthrindine, 7 quinoline alkaloids. Among them, five constituents were reported for the first time in Z. nitidum. In addition, a HPLC-MS/MS method was developed to simultaneous determination of three main absorbed components, including magnoflorine, α-allocryptopine, and skimmianine for the first time. This HPLC-MS/MS method was applied to pharmacokinetic study after oral administration Z. nitidum decoction. These results would be helpful for a better understanding about material basis and function mechanism of Z. nitidum decoction, and also provided important information for the quality control and further pharmacological study.