Chemical Profiling of Xueshuan Xinmaining Tablet by HPLC and UPLC-ESI-Q-TOF/MS

Xueshuan Xinmaining Tablet (XXT) is a widely used traditional Chinese medicine for the treatment of stroke, chest pain, coronary heart disease, and angina pectoris caused by blood stasis. Having a multiple-component preparation, it is still far from meeting the requirements of modernization and standardization because its detailed chemical basis and action mechanism have not been clarified. In this work, the different batches of XXT samples were analyzed by HPLC and the typical sample was analyzed by UPLC-ESI-Q-TOF/MS to understand its chemical profiling. As a result, 77 chromatographic peaks were detected, among which 63 constituents were identified or tentatively characterized 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 XXT products, which provides the sufficiently chemical evidence for the global quality evaluation of XXT products.

Ultraperformance liquid chromatography coupled with electrospray ionization tandem quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF/MS) has become one of the most effective tools for the on-line structural elucidation of multiple components of TCM, owing to its characteristics of accurate mass measurement, high resolution, and excellent sensitivity. MS E (E represents collision energy) technology provides an automated strategy to decrease 2 Evidence-Based Complementary and Alternative Medicine analysis time and maximize duty cycles by using parallel alternating scans at low collision energy in the collision cell to obtain precursor ion information or at high collision energy to obtain accurate full-scan mass fragment, precursor ion, and neutral loss information. Therefore, both precursor and fragmentation data in exact mass mode were collected in a single run. This method has been proved to provide excellent chromatographic and MS efficiencies in the previous publications [6]. In the present investigation, a combination of HPLC and UPLC-Q-TOF/MS analyses was employed to find out and identify the common chemical profile in various batches of XXT samples. A total of 63 constituents were identified or tentatively characterized based on the comparison with reference substances, on-line UV spectra, and the fragmentation rules. 110718-200507) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Gamabufotalin, bufotalin, and bufalin were purified from the fresh toad venom (the purity of gamabufotalin was over 92.9% and the purity of bufotalin and bufalin was over 98% based on the area normalization by HPLC analysis). Ginsenoside Rd, chlorogenic acid, rutin, quercetin, protocatechualdehyde, ursodesoxycholic acid, and chenodeoxycholic acid were from the collection of our laboratory and their purities were over 98%. The structures of all reference compounds were shown in Figure 1.

Materials and Methods
HPLC grade acetonitrile and formic acid (Fisher, Fair Lawn, NJ, USA) and ultrapure water were used. All other chemical reagents were of analytical grade from Beijing Chemical Corporation (Beijing, China).
. . Reference Solution Preparation. Stock solution with a concentration of about 0.1 g⋅L −1 was prepared by dissolving an accurately weighed amount of each reference substance in methanol.
. . Sample Preparation . . . XXT Sample. 1.0 g (about 2.5 tablets) of pulverized XXT sample was extracted with methanol (25 mL) by ultrasonication (200 w, 53 kHz) for 30 min at room temperature, and the extract was centrifuged for 5 min at 3000 rpm. The supernatant was evaporated at 80 ∘ C and the residue was dissolved with aqueous methanol (MeOH-H 2 O, 9:1). The obtained solution was centrifuged for 5 min at 12000 rpm. A volume of 10 L of the supernatant was used for HPLC analysis.
1 ml of the sample solution for HPLC analysis was diluted to a volume of 10 mL and used for UPLC-ESI-Q-TOF/MS analysis.
. . . Raw Materials. The ethanolic and aqueous extracts of individual herb (Danshen, Maodongqing, Shuizhi, Chuanxiong, and Huaihua) were prepared according to the manufacturing processes of XXT ( Figure S1) described in the current Chinese Pharmacopoeia [2]. The ethanolic and aqueous extracts along with Rengong Shexiang, Rengong Niuhuang, Renshen Jingye Zongzaogan, Bingpian, and Chansu were dissolved with aqueous methanol solution (MeOH-H 2 O, 9:1) according to the method described in Section 2.3.1 and used for the comparative analyses on the possible contribution from individual herb to general chromatographic profile of XXT samples.

. . Qualitative HPLC Analyses of Batches of XXT Samples.
The analyses were performed on a Shimadzu HPLC system (Shimadzu, Japan) equipped with an LC-20AT binary pump, a DGU-20A5 degasser, an SIL-20AC autosampler, a CTO-20AC column oven, and an SPD-M20A photodiode array detector. The samples were separated on a Waters XTerra C 18 column (4.6×250mm, 5 m). The mobile phase consisted of water containing 0.1% acetic acid (A) and acetonitrile (B) using a gradient elution program as follows: 0 min, 10% B; 20 min, 25% B; 30 min, 26% B; 45 min, 30% B; 60 min, 60% B; and 95 min, 80% B. The flow rate was 1.0 mL⋅min −1 and the column temperature was 30 ∘ C. The PDA detector recorded UV spectra in the range from 190 nm to 400 nm and HPLC chromatogram was monitored at 251 nm.
. . UPLC-ESI-Q-TOF/MS Analysis. To comprehensively identify the chemical constituents in XXT samples, a UPLC-ESI-Q-TOF/MS experiment was performed using a Waters Xevo G2-S spectrometer (Waters, America), connected to a Waters UPLC system (Waters, America). The samples were separated on a Waters ACQUITY BEH C 18 column (2.1×50 mm, 1.7 m). The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B) using a gradient elution program as follows: 0 min, 10% B; 4 min, 25% B; 7 min, 25.6% B; 8 min, 36% B; 14 min, 60% B; 18 min, 80% B; and 20 min, 80% B. The mass spectrometer was operated in both positive and negative modes using the following parameters: capillary voltage of 1.8 kV, sample cone voltage  Evidence-Based Complementary and Alternative Medicine of 30 V (ESI+) or 40 V (ESI-), source temperature of 120 ∘ C, desolvation temperature of 600 ∘ C, and cone gas flow 50 L/h. In MS E mode, the trap collision energy for the low-energy function was set at 6 eV, while the ramp trap collision energy for the high-energy function was set at 25-50 eV. Argon was used as the collision gas for collision-induced dissociation (CID) in MS E and MS 2 modes. To ensure mass accuracy and reproducibility, the mass spectrometer was calibrated over a range of 100-2000 Da for MS and 50-2000 Da for MS 2 . Leucine-enkephalin (m/z 556.2771 in positive ion mode; m/z 554.2615 in negative ion mode) was used as an external reference for the LockSpray and was infused at a constant flow of 5 L⋅min −1 . The data were recorded in centroid type during acquisition.

Results and Discussion
. . Qualitative Analyses of XXT Samples by HPLC. In order to obtain better detection of XXT samples, HPLC conditions were optimized. The acetonitrile-water system displayed more powerful separation ability for the main constituents in all samples than the methanol-water system. When organic acid was added to the mobile phase, the symmetry of most chromatographic peaks was improved. Under optimized conditions, ten batches of XXT samples, together with the reference compounds, were examined and their HPLC chromatograms were shown in Figure 2. High similarity in the number, type, and amount of chemical constituents was observed in the HPLC profile of each individual sample, which suggested the chemical consistency of different batches of XXT samples. The characteristic peak 4 (rutin) represents an important active component in XXT sample with a consistently high content, which was chosen as the reference peak for the calculation of the relative retention time ratio and relative peak area ratio of other common chromatographic peaks. For the validation of the assay procedure, the results of precision and repeatability were indicated by RSDs that were less than 4.9% (n=6) for seventeen peaks (Tables S1∼S4). The stability test suggested that these peaks were stable in the sample solution within 24 hr (Tables S5∼S6). To identify the origin of these characteristic peaks from individual raw material, a comparative study was carried out by using various extract of raw materials and XXT samples. Considering the chromatogram recorded at the wavelength of 251 nm, main chromatographic peaks in the HPLC profile were attributed to the following five raw materials: Danshen, Huaihua, Maodongqing, Chuanxiong, and Chansu ( Figure S2). The contribution of other raw materials was not manifested, either because of the absence of UV absorption of active compounds or due to the existence of no potent components at the detection condition. More information is expected to further confirm the contribution of each raw material to the general chromatographic profile of XXT sample.  extensive structural information via collision-induced dissociation. For the purpose of determining the optimized CE to generate fragment ions for structural elucidation and characterization, MS/MS experiments at different CE values were performed. Under optimized parameters, the total ion chromatogram of XXT samples are shown in Figure 3.

. . Identification of Chemical Constituents in XXT
The fragmentation behaviors of reference compounds were investigated, and these rules were applied for the structural elucidation of their derivatives with the same basic skeleton. A total of 63 compounds in XXT sample were detected and tentatively characterized by comparing the retention times, MS fragmentation behavior, and literature information. Among these compounds, there were twenty saponins, fifteen phenolic acids, ten quinones, eight steroids, four bile acids, four flavonoids, one amino acid, and one lactone. Their chemical structures are provided in Figure 1. For better understanding of the fragmentation behavior, 63 compounds were divided into several groups and deduced as follows. The chromatographic and mass data of the identified constituents were summarized in Table 1.    fragmentation ions were observed due to the neutral loss of glucose, rhamnose, arabinose, or xylose (see Figure 4). These fragmentation rules are in accordance with the previous data [7,8]. Totally, 20 ginsenoside saponins were identified, and they are deduced from the raw material, total ginsenoside of ginseng stems and leaves. Among the identified saponins, eight compounds (23, 24, 46, 47, 49, 53, 61, and 74) were unambiguously identified as ginsenoside Rg 1 , ginsenoside Re, ginsenoside Rb 2 , ginsenoside Rb 3 , 20S-ginsenoside F 1 , ginsenoside Rd, 20S-ginsenoside F 2 , and 20S-ginsenoside It could be one of ilexoside A or ilexoside D described in the literature [10].
Flavonoids. Flavonoids always present high sensitivity in the UV spectra. They could be well detected in both positive and negative ionization modes. Rutin, which has UV  [11].
Danshensu has UV max absorption at 279 nm and retention time at 1.03 min. It showed the precursor ion at m/z 197.0443 and the molecular formula was calculated as Bufadienolides. The MS/MS behaviors of bufadienolides have been extensively described [17,18]. Briefly, for bufadienolides with only hydroxyl substituents, the fragmentation was characterized by successive eliminations of H 2 O and CO molecules, and the profile of MS/MS product ions was correlated with the number of hydroxyl groups. If a C-16 acetoxyl group was present, the fragmentation of [M+H] + ions was triggered by initial loss of 60 Da (HOAc). The elimination of CO was significant for bufadienolides with a 19-formyl group, and the 19-hydroxyl group could be characterized by the loss of 30 Da (HCHO). These fragmentation rules were applied to the identification of bufadienolides in XXT sample. As shown in Table 1, eight bufadienolides were screened from XXT, six of which were unambiguously identified as gamabufotalin (19), arenobufagin (25), bufotalin (41), bufalin (50), resibufogenin (55), and cinobufagin (56) by comparison with reference substances isolated from toad venom. The other two bufadienolides were tentatively identified as bufarenogin (20) and hellebrigenin (29) [18,19]. Taking gamabufotalin as a case, the fragmentation rules of bufadienolides were explained ( Figure 5). Gamabufotalin showed UV max absorption at 295 nm. In its full mass spectrum, [ Quinones derivatives. Quinones derivatives are another kind of active constituents from Danshen and they were easily detected in XXT sample. Compounds 63, 71, and 76 were identified as dihydrotanshinone, cryptotanshinone, and tanshinone II A by comparison with reference substances. They displayed similar fragmentation pathways concerning successive eliminations of H 2 O and CO molecules. Tanshinone IIA was used as an example to illustrate the fragmentation pathway of quinones constituents as shown in Figure 6. Based on these rules as well as empirical molecular formula, 7 compounds were characterized as tanshindiol A 31, 1-oxo tanshinone IIA 59, neotanshinone D 62, tetrahydrotanshinone I 66, methyltanshinonate 69, methylenetanshinquinone 73, and miltirone 77. [12,16,20,21].
Phthalide derivative. Compound 21 has UV absorption at 276 nm. Its molecular formula was calculated as C 12 H 14 O 3 on the basis of [M+H] + ion at m/z 207.1024. The above data were in accordance with those of 4-hydroxyl-3-butylphthalide in the literature [22]. Compound 21 was tentatively assigned as 4-hydroxyl-3-butylphthalide. It could be derived from individual herb Chuanxiong rhizome [23].
Among the identified compounds, most constituents were derived from the raw materials, Danshen and Renshen Jingye Zongzaogan, and a small proportion of compounds were considered from Huaihua, Maodongqing, Chuanxiong, Chansu, and Rengong Niuhuang. The other ingredients, Rengong Shexiang, Bingpian, and Shuizhi, were not characterized in the present HPLC and UPLC-QTOF/MS condition. This could be related to the prescription of raw materials and the manufacturing process employed. Usually, muscone, one of active constituents in Rengong Shexiang and borneol are detected by GC or GC-MS [3]. They are not easily detected in the liquid chromatography. Additionally, it is worth noting that no chemical information of Shuizhi, a famous traditional Chinese medicine originated from animal source, was found, although small molecule compounds with diverse structures have been reported from this drug [24]. More effort is made to explain its prescription role in XXT product.

Conclusion
In this work, HPLC analysis was employed to find out the common chromatographic peak in various batches of XXT samples and UPLC-Q-TOF/MS was used for the identification of main constituents in the typical XXT sample. As a result, a total of 63 constituents including twenty saponins, four flavonoids, fifteen phenolic acids, eight steroids, four bile acids, ten quinones, and other two compounds were identified or tentatively characterized 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 clearly understood the nonvolatile constituents in XXT and provided good basis for further study on the active substances and quality control of this preparation.

Data Availability
HPLC and UPLC-ESI-Q-TOF/MS data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.