The Profiling and Identification of the Absorbed Constituents and Metabolites of Guizhi Decoction in Rat Plasma and Urine by Rapid Resolution Liquid Chromatography Combined with Quadrupole-Time-of-Flight Mass Spectrometry

Guizhi decoction (GZD), a well-known traditional Chinese medicine (TCM) prescription consisting of Ramulus Cinnamomi, Radix Paeoniae Alba, Radix Glycyrrhizae, Fructus Jujubae and Rhizoma Zingiberis Recens, is usually used for the treatment of common colds, influenza, and other pyretic conditions in the clinic. However, the absorbed ingredients and metabolic compounds of GZD have not been reported. In this paper, a method incorporating rapid resolution liquid chromatography (RRLC) with quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) was used to identify ingredients after oral administration of GZD. Identification of the primary components in GZD, drug-containing serum and urine samples was carried out in order to investigate the assimilation and metabolites of the decoction in vivo. By comparing the total ion chromatograms (TICs) of GZD, a total of 71 constituents were detected or characterized. By comparing TICs of blank and dosed rat plasma, a total of 15 constituents were detected and identified as prototypes according to their retention time (tR) and MS, MS/MS data. Based on this, neutral loss scans of 80 and 176 Da in samples of rat plasma and urine helped us to identify most of the metabolites. Results showed that the predominant metabolic pathways of (epi) catechin and gallic acid were sulfation, methylation, glucuronidation and dehydroxylation; the major metabolic pathways of flavone were hydrolysis, sulfation and glucuronidation. Furthermore, degradation, oxidation and ring fission were found to often occur in the metabolism process of GZD in vivo.


Introduction
Traditional Chinese medicine (TCM) prescriptions, usually made up of several medicinal herbals according to certain mass ratios guided by traditional Chinese medicine theory, have been used for centuries for the treatment of disorders or diseases in ancient and modern China [1][2][3]. It is well-accepted that a great deal of complex ingredients exist in compound Chinese traditional medicine (CCTM) and are effective through their preventive, therapeutic and synergistic effects [4,5].

Analysis the Constituents of GZD
In order to identify the constituents of Guizhi decoction (GZD), the rapid resolution liquid chromatography with quadrupole-time-of-flight mass spectrometry (RRLC-Q-TOF-MS) was used in positive and negative ion modes. Furthermore, the MS data including t R , values of m/z and MS/MS data was got from RRLC-Q-TOF-MS analysis. The total ion chromatograms (TICs) of GZD in electrospray ionization (ESI) negative and positive modes are listed in Figure 1.
The structures of most compounds in GZD were tentatively characterized by comparing their TOF-MS data, referring to related literature and comparing reference standards. Finally, a total of 71 compounds including 16 compounds originating from Radix Paeoniae Alba, one from Ramulus Cinnamomi, 33 from Radix Glycyrrhizae, four from Fructus Jujubae and 17 from Rhizoma Zingiberis Recens in GZD were detected by RRLC-Q-TOF-MS in negative or positive ion mode. In addition, 60 compounds of GZD had a known structure, and the others were isomers of part compounds from the explicit structures. The related information about identified components is listed in Table 1.

Analysis of Prototype Compounds in Plasma Sample
For the sake of analyzing the prototype components in plasma, we compared the TICs of dosed and blank rat blood. Compared with the normal control group, a total of 15 constituents were identified from rat plasma at dosed group according to their TOF-MS data (m/z, MS/MS, t R et al.). Among these 15 compounds, there are five compounds originating from Radix Paeoniae Alba (paeoniflorin, albiflorin, epicatechin, catechin and gallic acid), eight from Radix Glycyrrhizae (liquiritin, isoliquiritin, liquritigenin, isoliqurigenin, glycyrrhizic acid, naringenin-5-O-glucoside, naringenin-7-O-glucoside and formononetin), one from Ramulus Cinnamomi (cinnamic acid) and one from Rhizoma Zingiberis Recens (6-gingerol). The TICs of those 15 compounds and blank rat plasma are shown in Figure 2. In addition, prototype compounds of GZD including its related TOF-MS data can be found in Table 1.

Analysis of Metabolites in Blood and Urine Samples
As we know, after intragastric administration of the drug, the compounds originating from the drug were metabolized by intestinal bacteria in the intestine [16]. Then, they were absorbed into plasma so they can be metabolized further by all kinds of drug metabolism enzymes in liver. In principle, there are two metabolic reactions which are called phase I and phase II reactions. Through the phase I reactions including oxidation, reduction, and hydrolysis [17], the prototype components could be converted into aglycone, oxidized aglycone or reduced aglycone. After that, phase II reactions can convert the products of phase I into metabolites. In addition, the phase II reactions were focused on conjugating with glucuronide and sulfate [18][19][20][21]. In order to screen metabolites which were mainly conjugated with glucuronidation and sulfation, we use base peak chromatograms (BPCs) with neutral loss scans of 176 and 80 Da to find compounds existing in the rat plasma ( Figure 3a) and urine samples ( Figure 3b) in negative mode. A total of 47 peaks were identified which highly promoted the metabolite profiling of GZD. One metabolite was identified in positive mode. Comparing the TICs and, referring to references [22,23], another four metabolites were detected. The results showed that 52 components were tentatively detected as metabolites of GZD. All of the available information about the metabolites is shown in Tables 2 and 3. 2.3.1. Characterization of (epi) Catechin-Related Metabolites (Epi) catechin-related metabolites are the main metabolic constituents of Radix Paeoniae Alba. Referring to Liang's et al. researches [24] and comparing the mass spectrometry data, 21 constituents altogether were tentatively assigned in rat plasma and urine samples as metabolites, which were derived from (epi) catechin. The details are summarized in Table 2. The potential metabolism profile of (epi) catechin-related metabolites is presented in Figure 4.

Characterization of Gallic Acid-Related Metabolites
The metabolites of gallic acid-related are also the main metabolites of Radix Paeoniae Alba. By referring to Liang's et al. and Yan's et al. studies [24,25], glucuronidation and sulfation after deglycosylation was the principal metabolic pathway of gallic acid. Ten compounds altogether were identified in animal's plasma and urine samples as metabolites, which were derived from gallic acid. Among them, nine metabolites were characterized as glucuronide conjugates or sulfate conjugates. The available information about gallic acid-related metabolites is presented in Table 2, and a proposed metabolic pathway of gallic acid in rat is displayed in Figure 5.

Characterization of Flavone-Related Metabolites
The metabolites of flavone-related are the major metabolites from Radix Glycyrrhizae. There are different kinds of complicated flavone components such as naringenin-O-glucoside, liquiritigenin and fomononetin existing in GZD. Sixteen compounds altogether were assigned as flavone-related metabolites. Among them, six originated from naringenin-O-glucoside-related metabolites, six from liquiritigenin-related metabolites, three from isoliquiritigenin-related metabolites and one from fomononetin-related metabolites. The potential metabolism profile of the main flavone-related metabolites was described as shown in Figure 6, and information about all the flavone-related metabolites is listed in Table 2.

Characterization of Other Metabolites
Paeoniflorin-related, cinnamic acid-related and gingerol-related compounds were also the most important metabolites of GZD. There are five metabolites belonging to them, among which three originated from paeoniflorin-related metabolites, one from cinnamic acid-related metabolites and one from gingerol-related metabolites. Further details can be seen in Table 2.

Discussion
In present study, we mainly discussed the identification and detection of (epi) catechin-related, gallic acid-related and flavone-related metabolites as they were found to be significant metabolites with high content in GZD. In addition, other metabolites such as gingerol-related metabolites are briefly introduced. Compared to M1, catechin-O-glucuronide, M2 and M3 had an additional CH 2 unit according to their high resolution mass spectrometer (HRMS) data, which determined their formula to be C 22 H 24 O 12 . Hence, M2 and M3 were identified as Methyl (epi) catechin conjugating with glucuronide. Following a previously research [27], M2 and M3 were tentatively identified as 3'-O-methyl (epi) catechin 5-O-glucuronide and 3'-O-methyl (epi) catechin 7-or 4'-O-glucuronide, respectively. In the same way, another three (epi) catechin-related metabolites conjugating with glucuronide, M4, M6 and M20 were tentatively detected and identified as 5-(3,4-dihydroxyphenyl)-γ-valerolactone glucuronide, 5-(3-methoxyl-4-hydroxyphenyl)-valerolactone glucuronide and benzoyl glucuronide, respectively. M21 (t R = 10.089 min) was determined to be C 9 H 9 NO 4 according to the speculation of the HRMS data, and its displayed [M−H] − at m/z 194.0298. The main fragment ion at m/z 150.0562 showed a loss of 44 Da which indicated M21 might be an acid. According to a previous report [28], M21 was inferred to be 3-hydroxyhipuric acid. As for the (epi) catechin-related metabolites conjugating with sulfate, the neutral loss of molecular weight was 80 Da. According to this regulation, their HRMS data and related literature [29][30][31], we completely identified 14 compounds which were sulfate conjugates. Altogether, 21 constituents of (epi) catechin-related metabolites were identified in rat blood and urine samples.
M22 displayed [M−H] − at m/z 345.1354, and its ion fragmentation was predominant at m/z 169.1232 which made clear the elimination of glucuronide residue of M22. Combining with the HRMS data, M22 was considered as gallic acid glucuronide and its molecular formula was C 13 H 14 O 11 . Likewise, the identification of another eight metabolites was carried out, including two glucuronide conjugates (M24 and M28) and six sulfate conjugates (M25, M26, M27, M30 and M31). Isomeric compounds were also examined. For the sake of distinguishing between them, a comparison of their retention times and consultation with previous literature [25,32]  .0160, which not only indicated M37 and M38 were isomeric but also confirmed that both of them conjugate with glucuronide. Based on HRMS data and related literature [25,33], M37 and M38 were tentatively identified respective as naringenin-4'-O-glucuronide and naringenin-7-O-glucuronide, and their molecular formula was C 21 H 20 O 11 . M39 had major MS/MS ions at m/z 271.0350 and 150.9829, which indicated M39 was a naringenin-related compound. The neutral loss of 80 Da showed that M39 was a sulfate conjugate. Therefore, M39 was tentatively assigned as naringenin-O-sulfate. M36 had its main fragment ions at m/z 447.0951, 351.0192 and 271.0628. This information suggested that M36 was conjugating with both sulfate and glucuronide. In addition, it might be a naringenin-related compound. Therefore, M36 was assigned as naringenin-O-glucuronide-O-sulfate. Altogether, there were 16 components of flavone-related metabolites which were tentatively identified.
M51 showed [M+H] + at m/z 471.1015. Its major ion fragment was at m/z 295.0564 implying that M51 might be a compound conjugating glucuronide. Therefore, M51 was tentatively identified as 6-gingerol-O-glucuronide. Unfortunately, there are no other gingerol-related even if Rhizoma Zingiberis Recens-related metabolites were detected and identified. Considering there are many components of originated from Rhizoma Zingiberis Recens, the probable reason was that the main components of Rhizoma Zingiberis Recens were hard to metabolize. A previous report deemed that the major components of Rhizoma Zingiberis Recens are volatile oils which are not detectable and retainable with RRLC-Q-TOF-MS analysis [34]. The real reason actually remains unknown.

Reagents
Methanol and formic acid of HPLC grade were obtained from Merck (Darmstadt, Germany). Acetonitrile of HPLC grade was obtained from Fisher Scientific (Pittsburgh, PA, USA). Ultra-high purity water was prepared by Milli-Q system (Millipore, Billerica, MA, USA). All other chemicals were commercially available (Beijing Chemical Works, Beijing, China) and were of analytical reagent (AR) grade.
Radix Paeoniae Alba Ramulus Cinnamomi, Radix Glycyrrhizae, and Fructus Jujubae were obtained from Tong-Ren-Tang drugstore (Beijing, China). Rhizoma Zingiberis Recens was purchased from Dong-Zhi-Men vegetable market (Beijing, China). All of the crude drugs were identified by Chunsheng Liu (Academy of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China). The standards of Paeoniflorin, liquirituin, cinnamic acid, glycyrrhizic acid were got from National Institute for the Control of Pharmaceutical and Biological Products.

Rapid Resolution Liquid Chromatography with Quadrupole-Time-of-Flight Mass Spectrometry (RRLC-Q-TOF-MS) Analysis
RRLC-MS system is made up of an Agilent 1260 RRLC system coupled with Agilent 6520 Q-TOF mass spectrometer (6520, Aglient Technologies, Santa Clara, CA, USA). Both positive and negative ion modes were operated by an ESI source (6520, Aglient Technologies). Furthermore, full wavelength scanning analysis over an m/z range of 100-1500 was performed in positive or negative ionization mode. Data acquisition and processing were performed using Mass Hunter Qualitative Analysis B.04.00 software (Aglient Technologies).
The analytical column was operated by an Agilent ZORBAX SB-C18 (2.1 × 50 mm, 1.8 µm). The mobile phase is made up of 0.1% formic acid water (A) and acetonitrile (B). The linear elution gradient was as follows: 0-5 min, 5% B; 5-15 min, 5%-25% B; 15-25 min, 25%-40% B; 25-45 min, 40%-95% B. The injection volume was 2 mL. The flow rate was 0.3 mL/min and column temperature was at 35 • C. The conditions of the mass spectrometer under the ESI mode were as follows: ion spray voltage was 3500 V; N 2 as drying gas and its flow rate was 10 L/min. The temperature of N 2 350 • C; the pressure of nebulizer was 40 spi. The collision energy was set at 20 V in initial and then changed when necessary.

Preparation of Guizhi Decoction (GZD)
To prepare the GZD, the crude drugs-namely, Ramulus Cinnamomi (9 g), Radix Paeoniae Alba (9 g), Radix Glycyrrhizae (6 g), Rhizoma Zingiberis Recens (9 g) and Fructus Jujubae (12 pieces)-were immersed in an eight-fold mass of distilled water for 30 min. After that, the mixture decocted for 30 min and filtered. Subsequently, a six-fold mass of distilled water was added to the mixture and decocted for another 30 min. We were combining the two filtrates and making the concentration of crude drug to 1 g/L.

Animal Housing Environment and Experiments
Fourteen healthy male Sprague-Dawley (SD) rats (200 ± 20 g) used in the experiments were purchased from Vital River Laboratory Animal Co., Ltd. (Beijing, China). They were maintained under standard conditions with light cycles of 12 h on and 12 h off. The room temperature and relative humidity were 23 ± 3 • C and 50% ± 10%, respectively. Principles of laboratory animal care and all protocols were in accordance with the relevant national legislation and local guidelines and were approved by Animal Care and Use Committee of the Institute of Basic Theory for Chinese Medicine, China Academy of Chinese Medical Sciences (Date: 6 August 2014; No.: 201408-16).
After 7 days' acclimation in the metabolism cages and 12 h fasting with only water ad libitum, animals were used in experiments and randomly divided into two groups. Rats were orally administrated with GZD (10 mL/kg) once a day for two consecutive days as experimental group in Group 1. Rats were orally administrated with an equivalent volume of distilled water as normal control in Group 2. Then, 24 h urine was collected on the 9th day.
On the 9th day after the final oral administration, all rats were anaesthetized with 4% chloral hydrate, and the blood samples were collected from the portal vein. The animal serum of the same group was combined. The serum samples were obtained by centrifugation of blood at 3500 rpm for 15 min and were kept frozen at −80 • C until necessary.

Preparation of GZD
To prepare the test products of GZD, GZD (prepared before, 1 g crude drug per milliliter) of 0.1 mL were placed in 10 mL volumetric flasks, diluted with methanol to volume and filtered with 0.22 µm PTFE membrane.

Preparation of Plasma Samples
Two milliliter plasma was spiked with 6 mL methanol by vortex mixing for 30 s. Then, the mixture was immediately centrifuged for 15 min at 3500 rpm and at 4 • C to obtain the supernatant. The supernatant was shifted and evaporated to dryness. Then the residue was dissolved in 1 mL methanol. After that, the reconstituted extraction was centrifuged again for 15 min at 12,000 rpm and at 4 • C. At last, the supernatants were stored at 4 • C until RRLC-Q-TOF-MS analysis.

Preparation of Urine Samples
Urine samples were evaporated at 65 • C to dryness. The residues were dissolved in 10 mL methanol, and the reconstituted extraction was centrifuged for 15 min at 3500 rpm and at 4 • C to obtain the supernatant. The supernatant was transferred and evaporated to dryness. Then, the residues were thoroughly dissolved in 5 mL methanol. After that, the mixture was filtered with 0.22 µm PTFE membrane. Finally, the filtrates were stored at 4 • C until RRLC-Q-TOF-MS analysis.

Conclusions
In this paper, an efficient RRLC-Q-TOF-MS method was used for separation and identification of absorbed constituents and metabolites in rat blood and urine after gavaging GZD. Altogether, 67 constituents comprising 15 prototype compounds and 52 metabolites were detected and tentatively identified in rat urine and plasma samples. In addition, 71 components altogether originating from GZD were detected or characterized. The results showed that phenolic compounds such as gingerol and shogaol were the main constituents of Rhizoma Zingiberis Recens in GZD. Cinnamic acid was the major compound of Ramulus Cinnamomi in GZD. Both phenolic compounds and cinnamic acid were found to be small polor compounds, which can be quickly absorbed into plasma. This feature indicated that GZD can be used for clinical treatment of exterior syndromes such as common colds and pyretic conditions. The major constituents of Radix Paeoniae Alba, Radix Glycyrrhizae and Fructus Jujubae were flavone and saponin. Most of them were absorbed into plasma and were metabolized by all kinds of metabolism enzymes in liver. After two-phase reaction in the liver, they were metabolized to secondary metabolites, which took effect in vivo. Analysis of metabolites showed that (epi) catechin, gallic acid and flavone were the major sources of metabolites, which originated from metabolism of GZD in vivo. Sulfation and glucuronidation were the main metabolic pathways in the metabolic process of GZD in vivo. This study systematically explored the plasma and urine metabolic profiles of GZD. The results of this study can offer essential data for deeper pharmacological and clinical studies in GZD.