Chemical Profiling of an Antimigraine Herbal Preparation, Tianshu Capsule, Based on the Combination of HPLC, LC-DAD-MSn, and LC-DAD-ESI-IT-TOF/MS Analyses

Chemical profiling is always the first task in the standardization and modernization of Traditional Chinese Medicine. HPLC and LC-MS were employed to find out the common chromatographic peaks in various batches of Tianshu Capsule (TSC) and the contribution of the characteristic peaks from individual herbs to the whole chromatographic profile of TSC sample. A total of 38 constituents were identified in TSC sample based on the comparison of retention time and UV spectra with authentic compounds as well as by summarized MS fragmentation rules and matching of empirical molecular formula with those of published components. This is the first systematic report on the chemical profiling of the commercial TSC product, which provides the sufficiently chemical evidence for the global quality evaluation of TSC products.


Introduction
Traditional Chinese Medicine (TCM) is getting more attention all over the world due to its exact clinical practice, especially prescription application, which comprehensively highlights the quintessence of the theory of traditional Chinese medical science. Da Chuan Xiong Fang (DCXF), a well-known and extensively used TCM decoction for the treatment of migraine, first appeared in Xuan Ming Lun Fang, a famous formula book written by Wansu Liu who lived in Jin Dynasty (1115-1234). It is composed of two herbs, namely, Chuanxiong (Chuanxiong rhizoma) and Tianma (Gastrodiae rhizoma), with a crude weight ratio of 4 : 1. Eight dosage forms of DCXF such as capsule, tablet, dripping-pill, honeyed pill, oral liquid, and granule, have been authorized to Chinese market. Tianshu Capsule (TSC), as a representative of DCXF preparations, is widely used in clinics for treating the blood stasis type of headache and migraine [1,2].
Phytochemical and pharmacological investigations showed that phenols, organic acids, phthalides, and nitrogen-containing compounds were the major active ingredients of DCXF [3]. Several qualitative analyses have been reported concerning main types of constituents in DCXF [4][5][6][7]. One study described the identification of 17 different constituents in the 50% EtOH extract of DCXF by LC-Q-TOF/MS, containing gastrodin, parishin C, ferulic acid, guanosine, adenosine, palmitic acid, and 11 phthalide compounds [4]. Another similar study identified 3 compounds of Chuanxiong (ferulic acid, senkyunolide I, and senkyunolide H) and 8 constituents of Tianma (gastrodin, s-(4-hydroxybenzyl)-glutathione, parishin, parishin B, parishin C, p-hydroxybenzaldehyde, etc.) by using the HPLC-DAD-MS n coupling technique, respectively [5]. Continuous reports from the second research group confirmed that 10 different compounds, including 6 original substances of Chuanxiong and 4 original ones of Tianma, 2 Evidence-Based Complementary and Alternative Medicine were detected in the rat plasma after the gavage of DCXF active components [6,7]. However, all these investigations mentioned above were carried out based on the samples of the 50% EtOH extract of the mixture of both herbs (4 : 1) or active components of single crude herb. No systematic reports could be available involving the chemical profiling of the commercial finished products derived from DCXF. TSC was produced from both crude herbs by employing the various pharmaceutical engineering technologies and the complex manufacturing processes such as extraction, concentration, and preparation. The accumulating studies showed that decocting could induce chemical changes of medicinal herbs or combinatorial formula [8]. It is wellknown that ferulic acid and some of the phthalides such as Z-ligustilide and dimeric phthalide are unstable at high temperature [9,10]. Therefore, during the preparation of TSC, these thermolabile components may undertake chemical transformation, consequently leading to the difference of chemical compositions of finished products with DCXF decoction. Understanding the chemical profiles of TSC samples would be helpful in selecting suitable chemical markers for the quality control and pharmacokinetic study. In this work, a combination of HPLC, LC-DAD-MS n , and LC-DAD-ESI-IT-TOF/MS analyses was employed to find out and identify the common components in various batches of commercial TSC samples. The contribution of the characteristic peaks from individual herb to the whole chemical profiling of TSC was also discussed. A total of 38 constituents were identified or tentatively characterized, among which the water-soluble compounds with higher polarity from Gastrodiae rhizoma are detected in TSC samples for the first time.

Qualitative HPLC Analyses of 5 Batches of TSC Samples.
The analyses were performed on a Shimadzu HPLC system (Shimadzu, Japan) equipped with a LC-20AT binary pump, a DGU-20A5 degasser, a SIL-20AC autosampler, a CTO-20AC column oven, and a SPD-M20A photodiode array detector. The samples were separated on a Phenomenex Luna C 18 column (5 m, 4.6 × 250 mm). The mobile phase consisted of methanol (A) and water containing 0.1% formic acid (B) using a gradient program as follows: 0 min, 15% A; 5 min, 15% A; 55 min, 95% A; 60 min, 95% A. The flow rate was 1.0 mL⋅min −1 and the column temperature was set at 30 ∘ C. The PDA detector recorded UV spectra in the range from 190 nm to 400 nm and HPLC chromatogram was monitored at 276 nm.

Comparison of Typical TSC Sample and Its Related Crude Herbal Materials as Well as DCXF by HPLC and LC-MS.
TSC sample and its related crude herbal material, Chuanxiong rhizoma and Gastrodiae rhizoma as well as DXCF, were analyzed under the same chromatographic conditions by HPLC and LC-MS to find the contribution of individual herb to the whole chemical profile of TSC sample. The HPLC system was the same as those in Section 2.3. LC-MS analyses were performed using an Agilent 6130 Quadrupole LC-MS (Agilent, Waldbronn, Germany) connected to an Agilent 1200 HPLC system (Agilent, Waldbronn, Germany). The parameters for MS analysis in the positive and negative ion mode were as follows: nebulizer, 35 psi; ionization voltage, 3500 V; dry temperature, 350 ∘ C; flow rate of carrier gas, 9.0 L⋅min −1 . Full-scan mass spectra were acquired in the range of 100-800 m/z.

LC-DAD-ESI-IT-MS Analysis of Typical TSC Sample.
To comprehensively identify the chemical constituents in    Evidence-Based Complementary and Alternative Medicine TSC sample by the fragmentation rules, a LC-DAD-ESI-IT-MS n experiment was performed using an Agilent 6320 ion-trap spectrometer (Agilent, Waldbronn, Germany) connected to an Agilent 1200 HPLC system (Agilent, Waldbronn, Germany). The HPLC conditions were the same as those described in Section 2.3. The LC effluent was introduced into an electrospray ionization source after a postcolumn split ratio of 2 : 1. The parameters for MS analysis in the positive ion mode were as follows: nebulizer, 45 psi; ionization voltage, 4000 V; dry temperature, 350 ∘ C; flow rate of carrier gas, 12.0 L⋅min −1 . Full-scan mass spectra were acquired in the range of 100-800 m/z. The optimized parameters for MS/MS analysis were as follows: collision energy, 1.5 V; nitrogen was used as the collision gas. MS spectra of pure substances were obtained using the same parameters as mentioned above. The ultrahigh purity argon was used as the collision gas for collision-induced dissociation (CID) experiments, and the collision energy was set at 50% for MS 2 and MS 3 ; ion accumulated time was 30 ms. The MS data were collected in an automatic mode and the software could automatically select precursor ions for MS analysis according to criteria settings. Accurate mass determination was corrected using the external standard method. The data acquisition and analysis were performed by LC-MS Solution Version 3.6 software (Shimadzu, Kyoto, Japan).

Qualitative Analyses of TSC and Its Related Crude Herbal Materials by HPLC and LC-MS.
Under the HPLC conditions as described in the current Chinese Pharmacopoeia [1], 5 batches of TSC samples, together with the reference compounds, were examined and their HPLC chromatograms were shown in Figure S2. High similarity in the number, type, and amount of chemical constituents was observed in the HPLC profiles of different batches of TSC samples. General chromatographic profile was obtained by Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine software and characteristic peaks were found in the HPLC profile of each individual sample. In order to identify the origin of these characteristic peaks from individual herbs, a comparative study was carried out by using various extracts of herbs and TSC samples. Accordingly, the possible individual contribution from the corresponding herbs to the general chromatographic profile was found. Compared with the HPLC profiles of the ethanolic and aqueous extracts of Chuanxiong rhizoma and Gastrodiae rhizoma ( Figure S3 (Table 1).

Identification of Chemical Constituents in TSC by LC-DAD-EST-IT-MS and LC-DAD-ESI-IT-TOF/MS. The combination of LC-DAD-ESI-IT-MS and LC-DAD-ESI-IT-
TOF/MS experiments was employed for the identification of chemical constituents in TSC sample, and, as a result, a total of 38 compounds was identified or tentatively characterized. The structures of the identified compounds are shown in Figure 1 and their chromatographic and mass spectrometric data are shown in Tables 2 and 3. The total ion chromatograms (TICs) of TSC sample are shown in Figures S11 and S12, respectively. Among the identified constituents, 7 compounds (2, 3, 5, 6, 7, 9, and 26) were unambiguously identified as gastrodin, 5-HMF, parishin B, parishin C, parishin, ferulic acid, and Z-ligustilide based on the direct comparison of their retention times, UV spectra, and mass spectra with those of the authentic compounds. Furthermore, 31 compounds were tentatively characterized according to their UV spectra, empirical molecular formula, and mass fragmentation pathways as well as their eluted sequence from ODS column reported in the literature [11][12][13][14] and acquired in the present experiments.

Fragmentation Characteristic of Reference Compounds.
In the present HPLC and MS conditions, characteristic MS adduct ions were observed for phenolic glycosides, organic acid, and phthalide derivatives. Phenolic glycosides and organic acids could be well-detected in positive and negative ionization mode and adduct ions such as Da was assigned to p-hydroxybenzyl alcohol just like the characteristic loss of gastrodin. Therefore, compound 1 was identified as elatoside, namely, 6 -(p-hydroxybenzyl methyl)-gastrabiose, which was previously isolated from the rhizome of G. elata [16].     [15], whereas 12 showed elimination of two H 2 O followed by side-chain cleavage. Therefore, 11 was deduced as 4,5-dihydro-3,1 -dihydroxy-3-butylphthalide and 12 could be one of senkyunolide I and senkyunolide H [14,15] [14], they were tentatively identified as riligustilide, 3 ,6,8 ,3a-biligustilide, tokinolide B, levistolide A, and senkyunolide O, respectively. Three compounds, indicating the protonated ion [M+H] + at m/z 383, were detected in extracted ion chromatograms; however, only senkyunolide P was previously reported in Chuanxiong Rhizoma. Thus, compound 30 was tentatively characterized as senkyunolide P and the other compounds (33 and 38) were still indefinite based on the available information.
In the modernization of TCM, chemical profiling is always the first task. It is of importance for development of the suitable quality standard and control strategy, study of pharmacokinetics, and interpretation of therapeutic character of TCM [19,20]. As a marketed product in China, the quality control system of TSC is still to be improved and its pharmacokinetics and action mechanism are not completely clear. Usually, gastrodin, ferulic acid, and 6,7dihydroxyligustilide are selected as marker compounds for the quality control or the pharmacokinetic study of Tianshu Capsule [1,21,22]. The present investigation provides more chemical information for the selection of the marker compounds and the improvement of quality control. It also tells the pharmacokinetic scientists and ethnopharmacologists which compounds in TSC samples are worth further evaluation. In addition to gastrodin and ferulic acid, more efforts should be made to the group of phthalide derivatives such as major constituents, senkyunolide I/H 12, 4-hydroxy-3-butylphthalide 13, senkyunolide A 20, and Z-ligustilide 26 as well as their dimers. Additionally, it is worth noting that 5-HMF 3, a potent toxic compound, was found as main peak in the chemical profile of TSC samples. This compound originated from the crude material Gastrodiae rhizoma and its content in TSC samples was up to 2.67 mg⋅g −1 [23]. If it is necessary to develop its limit standard in TSC product or not, more study are expected. Also, ligustrazine is not detected in TSC sample, even in the extracted ion chromatogram, which could result from its very low amount in crude herbal material (about 0.01∼0.02%), although, actually, it is often considered as one of active components in Chuanxiong rhizoma.

Conclusions
In this study, HPLC analysis was employed to find out the common chromatographic peak in various batches of TSC samples. The contribution of the characteristic peaks from individual herbs to the whole chromatographic profile was discussed based on comparative HPLC and LC-MS analyses. A total of 38 constituents were identified based on the comparison of retention time and UV spectra with authentic compounds as well as by summarized MS fragmentation rules and matching empirical molecular formula with those of published components. The present investigation provided the good basis for monitoring the manufacturing processes and improving quality control of TSC products.