Chemical Fingerprint and Metabolic Profile Analysis of Tianshu Tablets by Ultra-High Performance Liquid Chromatography/Quadrupole-Time of Flight Mass Spectrometry

In recent years, the chemical fingerprinting of traditional Chinese medicines and the metabolites in these compounds has been a hot topic. In the present study, the chemical fingerprint of Tianshu tablets (TST) and the metabolic characteristics of compounds in rats after intragastric administration were studied by ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC/Q-TOF MS). In a preliminary study, 77 chemical components in TST were determined by comparison with retention times, accurate molecular mass, and characteristic fragment ions of the known compounds in the literature and some well-known compounds were analyzed in detail, and the fragmentation pathways for parishins B, gastrodin A, and cnidilide or neocnilide were specifically analyzed. After intragastric administration of TST (4 g/kg) to rats, a total of 61 compounds were detected in plasma samples, including 7 prototypes and 54 metabolites. After further analysis, it was found that these metabolites were subjected to glucuronidation, sulfation, methylation, hydroxylation, dehydrogenation, or mixed metabolic processes. Hydroxylation and glucuronidation were finally confirmed as the main metabolic pathways. This is the first research on the chemical fingerprint and metabolites of TST, which lays a foundation for further investigation of TST.


Introduction
In recent years, traditional Chinese medicine (TCM) has attracted increasing attention worldwide by virtue of its applications. Da Chuanxiong Formula (DCXF) is a wellknown and extensively used traditional Chinese medicine (TCM) decoction for the treatment of migraine caused by blood stasis and wind-heat syndrome. It is composed of two herbs, namely, Chuanxiong (Chuanxiong rhizoma) and Tianma (Gastrodiae rhizoma), with a crude weight ratio of 4 : 1. Tianshu tablets (TST) are a representative DCXF preparation that is widely used in clinics for treating the blood stasis type of headache and migraine [1][2][3].
Phytochemical and pharmacological investigations of DXCF have shown that phenols, organic acids, phthalides, and nitrogen-containing compounds are the major active ingredients [4]. At present, several qualitative studies on the main components of DCXF have been performed [5][6][7][8]. One study used LC-Q-TOF/MS to identify 17 different components in a 50% ethanol extract of DCXF [5]. In one study, three compounds of Chuanxiong and eight components of Tianma were identified by HPLC-DAD-MS n [6]. Two continuous studies showed that 10 different compounds were detected in rat plasma after intragastric administration of DCXF active components, including 6 compounds from Chuanxiong and 4 compounds from Tianma [7,8]. ese four studies were based on samples of a 50% ethanol extract from a 4 : 1 mixture of the two herbs or active ingredients from a single crude herb. In one study, 38 components were identified or preliminarily identified from a Tianshu capsule by means of HPLC, LC-DAD-MSN, and LC-DAD-ESI IT-TOF/MS analysis, although Tianshu tablets and Tianshu capsules are two different dosage forms [9]. is research is still very important because of its different applicability. is study enriches our understanding of the components of DCXF and studies the metabolites of TST for the first time.
In our study, 77 chemical components of TST were preliminarily determined by a comparison with retention time, accurate molecular mass, and characteristic fragment ions of known compounds in the literature. Furthermore, UPLC/Q-TOF MS was used to analyze the plasma of rats after oral administration of TST. A total of 61 compounds were identified or preliminarily identified, including 7 prototypes and 54 metabolites.

Chemicals and Materials.
Some reference standards (pyroglutamic acid, 5-(hydroxymethyl)furoic acid, and parishin B) for Gastrodia rhizoma were isolated and purified in our laboratory, and other standards (uridine, gastrodin A, and neocnilide) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Purities of the standards were above 98% by HPLC analysis. HPLC-grade acetonitrile, methanol, and formic acid were purchased from Fisher Scientific (MA, USA). Deionized water was prepared by a Milli-Q Water purification system (Millipore, MA, USA). High-purity nitrogen (99.99%) and helium (99.999%) were purchased from Gas Supplies Center of Peking University Health Science Center (Beijing, China).
Gastrodia rhizoma and Chuanxiong rhizoma were purchased from Tian Heng pharmacy (Beijing, China). All herbal materials were authenticated by Professor Bei Wu (Nanchang Institute for Food and Drug Control). TianShu tablets were prepared according to Chinese Pharmacopoeia 2015 Edition [1].

Animals and Drug.
Sprague-Dawley rats (male, 12-14 weeks; 200-240 g) were provided by Hunan SJA Laboratory Animal Co., Ltd. Protocols for all animal experiments were approved. Animals were kept in a controlled environment for 3 days and fasted for 12 h before experiments. TST were dissolved in a 9 g/L NaCl solution (NS) (250 mg/ml) and administered by oral gavage at a dose of 1000 mg/kg (equivalent to 4 g of crude drug per kg) body weight.

Sample Collection and Pretreatment.
After oral administration of TST, blood samples were collected at 30, 60, and 120 min (n � 5) in an Eppendorf tube with heparin sodium and then centrifuged (16000 rpm) at 4°C for 10 min. e supernatant was then separated, and all samples were stored at − 80°C immediately until analysis. e protocol for sample preparation is described below: 1 mL plasma was mixed with 5 mL methanol, vortexed for 5 min, and centrifuged at 16000 rpm at 4°C for 20 min. e supernatant was dried with nitrogen gas at 4°C, and 400 μl of 50% methanol was added to reconstitute the residue, which was then centrifuged at 16000 rpm for 10 min at 4°C. e supernatant was transferred to a vial, and 10 μl was injected for LC-MS analysis. All samples were filtered through a membrane (0.22 μm pore size). At the same time, in order to eliminate the influence of matrix, blank plasma was added to participate in the analysis. e conditions for the ion source were as follows: compounds in TST were measured using the total ion chromatograms in negative and positive ion ESI-MS mode in the mass range m/z 50-1250, but plasma samples were analyzed only in positive ion ESI-MS mode. e other operating parameters were optimized as follows: source temperature, 500°C; ion spray voltage, 4500 V; gas 1, 50 psi; gas 2, 50 psi; curtain gas, 45 psi; decluttering potential, 100 V; and collision energy was set to 40 (15) eV.

Data
Process. TST compounds from the extracts and metabolites data were acquired by full scan, which rely on dynamic background subtraction (DBS) and multiple mass defect filtering (MMDF) and includes some compounds with very low concentrations (MDF window was set to ±50 mDa around the mass defects of the templates and over a mass range of ±50 Da around the filter template masses).
Analysis of data on TST compounds in extracts and metabolites was performed using a variety of data mining tools, including extract ion chromatograms (XIC) of compounds in TST could be separated within 48 minutes, while plasma samples could be separated within 30 minutes (the specific methods can be found in Section 2.4). In addition, in order to obtain the most abundant mass spectrometry information, the collision energy was optimized. e results showed that when the collision energy rose to 40 eV, the main fragments were seen, but when the energy reached 55 eV, the second order fragments were too fragmented to be easily analyzed. erefore, a collision energy of 40 eV was selected. As for UPLC/Q-TOF MS, mass spectra were recorded in both positive and negative detection modes.

UPLC/Q-TOF MS Analysis of TST Extracts.
To characterize the chemical constituents of TST, a fast, efficient, and reliable UPLC/Q-TOF MS method was established. By virtue of the high resolution and speed of UPLC and the accurate mass measurement of the TOF MS, a total of 77 compounds were identified. e mass spectra of these components were examined in negative ion mode and positive ion mode. e total ion chromatogram (TIC) of TST in positive and negative ion modes are shown in Figures 1 and 2. Details of the identified components are summarized in Table 1. rough analysis, it was found that the 77 compounds contained 19 organic acids, 9 nitrogen-containing compounds, 11 glucosides, 8 phenols, 24 phthalides, and 6 other compounds. e numbering information of these compounds is shown in Figure 3.

Chemical Fingerprint of TST in Negative Ion Modes.
According to the literature, the main components of Tianma are phenols and organic acids [9]. However, there are also glycosides in the components of Tianma [12]. Many characteristic components of Tianma were analyzed and identified in the negative ion mode. Because the structures of organic acids and phenols are relatively simple, the characteristic glycoside compounds X18 and X23 were identified here and the chromatographic and spectral data for compounds X18 and X23 were preliminarily characterized by referring to the literature and reference materials. Peak

Chemical Fingerprint of TST in Positive Ion Modes.
e analysis of the positive ion mode results showed that the characteristic components of Chuanxiong, including phthalides, were present. Here, compound Y27 was selected for analysis, and the chromatographic and spectral data of this compound were analyzed by comparison with the literature and reference materials. e cleavage pathway of phenyl peptides in Chuanxiong was also analyzed.
Peak Y27 gave a [M+H] + ion at m/z 195.1378 and fragment ions at m/z 177.1344, 149.1309, and 107.0550. According to previous literature reports [9] and a reference standard, we identified peak Y27 as cnidilide or neocnilide. e characteristic fragmentation pattern of Y27 is shown in Figure 4(c) According to our analysis, the main components of Tianma in negative ion mode were organic acids, phenols, and glycosides, with mainly phthalides detected in positive ion mode. e specific pyrolysis fragments were similar to the standards.

Detection and Identification of the Metabolites of TST in
Rat Plasma. In order to identify as many potential pharmacologically active compounds as possible in TST, metabolic profiling of TST in rat plasma was performed. Compounds absorbed in vivo can be further metabolized by a variety of enzymes through oxidation, hydrolyzation, methylation, glucuronidation, and sulfation. Only peaks that were detected in the dosed plasma samples but not in blank samples were considered as probable metabolites. e mass spectra of the metabolites were examined in positive ion mode. ese were further analyzed by using Peakview 1.2 to identify expected and unexpected metabolites from different metabolic pathways, and their structures were identified by tandem MS. We selected senkyunolide D or 4,7-dihydroxy-3-butylphthalide and senkyunolide A as examples of the structural identification process. e metabolites of these compounds and others are summarized in Table 2  Intensity Time (min)       Journal of Analytical Methods in Chemistry  a "X" in negative ion mode and "Y" in negative-positive mode. b Compared with reference standards.    Journal of Analytical Methods in Chemistry erefore, M5 might be a metabolite of senkyunolide D after hydrogenation and glucuronidation, while M6 might be a metabolite of senkyunolide D after hydroxylation and glucuronidation (metabolites of M1 and extract ion chromatograms (EICs) are shown in Figure 6).
Metabolite M27, which eluted at 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Time (  A, suggesting that M32 might be a metabolite of senkyunolide A after H 2 O conjugation. Metabolite M33, which eluted at 9.56 min, formed a molecular ion of [M+H] + at m/ z 225.1119 corresponding to C 12 H 16 O 4 . Its major fragment ions m/z 207.1013 and 189.0914 were generated by loss of 18 Da and 18 + 18 Da, which implied loss of one or two H 2 O groups. Other product ions were identical to that of M27. erefore, M33 might be a metabolite of senkyunolide A after 2 hydroxylation events (metabolites of M27 and extracted ion chromatograms (EICs) are shown in Figure 7).
Sixty-one metabolites were identified in rat plasma. rough the analysis of these 61 metabolites, it was found that hydroxylation and glucuronidation were the main metabolic ways following oral administration of TST. From the identified metabolites, it can be speculated that after absorption of TST by human blood, most of the compounds undergo hydroxylation and glucuronidation, which allow TST to play a positive role in the treatment of migraine and blood stasis headaches. is provides a basis for follow-up research on the medical uses of TST. At the same time, from the information obtained on the metabolites, it can be seen that the main metabolites in positive ion mode of TST are concentrated as chuanxiong lactones, but there are no effective metabolites from Tianma. It is possible that Tianma metabolites are mainly present in the negative ion mode of plasma or in feces, urine, and bile, which requires further study.

Conclusion
In this study, UPLC/Q-TOF MS was used to comprehensively determine the chemical fingerprint and metabolic profile of TST after intragastric administration. In the analysis of the chemical constituents of TST, 77 compounds were identified, including 39 compounds identified in negative ion mode and 38 compounds identified in positive ion mode. In order to elucidate the mass spectrometric pyrolysis law of the main compounds in TST, gastrodin A, parishin B, and cnidilide or neocnilide were specifically analyzed, and the results were completely consistent with the results in reference standards and the reported literature. And 61 metabolites of TST in rat plasma were detected, which were mainly metabolites of 7 compounds. Two prototypes (senkyunolide D or 4,7-dihydroxy-3-butylphthalide and senkyunolide A) and their metabolites were analyzed in detail, which showed hydroxylation and glucuronidation were the main metabolic pathways following oral administration. is study expanded our understanding of the chemical constituents of TST, studied its metabolic spectrum for the first time, and clarified its main metabolic pathway in plasma, which will lay the foundation for follow-up studies of the pharmacological mechanism of TST.

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

Conflicts of Interest
e authors declare no conflicts of interest in publication of this study.