Analysis of Chemical Constituents of Guizhi Shaoyao Zhimu Granules by UPLC-Q-Orbitrap HRMS


 Background: Guizhi Shaoyao Zhimu Granules (GSZG), which are derived from Guizhi Shaoyao Zhimu decoction in China, have good clinical efficacy in Rheumatoid arthritis(RA) especially. However, there is currently no reliable and systematic method to research the effective ingredients of the kind of prescription. Methods: Vanquish Ultra High Performance Liquid Chromatography Q Exactive IV Pole-electrostatic field orbital trap high resolution mass spectrometer (UPLC-Q-Orbitrap HRMS), a quick and systematic method was established and apply to analysis the chemical component of GSZG. According to using reference standards comparsion, fragmentation rules elucidation and available databases like mzCloud and the OTCML to identify the components of GSZG.Results: The results showed that it identified a total of 74 components, including 2 chromone, 23 flavonoids, 10 alkaloid, 9 phenolic acid, 8 nucleosides and nucleobases, and 22 other components, were characterized from GSZG. Conclusion: this was the first time to systematically report the compounds of GSZG, the chemical basis inquiry of this work would have a profound impact on mechanism exploration and providing certain reference for the preparation and quality control of GSZG developed by China independently.

Preparation of sample solutions GSZG (0.5g) was put into a 50ml Erlenmeyer ask respectively after weighing accurately, added 25ml of 75% methanol, weighed, extracted using an ultrasonic bath (40kHz, 150W) for 30 minutes, cooled, adjusted to make the weight the same as before, shake well, and nally ltered each sample solution with a 0.22μm microporous membrane before being injected into UPLC instrument for analysis.

Instrument and analysis conditions
The experiments were performed on Vanquish Ultra High Performance Liquid Chromatography Q Exactive IV Pole-electrostatic eld orbital trap high resolution mass spectrometer(Thermo Fisher Scienti c, USA). Xcalibur software (version 3.0) was used to control instrument, collect data and analysis data.
The spray voltage is 3.2kV, the ion source temperature is 350°C, the sheath gas ow rate is 35arb, the auxiliary gas ow rate is 10arb, and the ion transfer tube temperature is 320°C. The compound was scanned by using a full scan/data-dependent secondary scan method, the primary resolution was 70,000, the secondary resolution was 17,500, the scanning range from m/z=100 to m/z=1500, and the collision energy gradient was 20ev, 40ev, and 60ev.

Data processing
The collected raw data was imported into Compound Discoverer 3.0 software and MZmine (version 2.53) software [19], and the identi cation process of unknown compounds was established through its wizard settings and method templates. The software was performed peak alignment and peak extraction on the original data, so that the obtained molecular ion peaks, possible molecular formulas tted by isotope, and the measured spectra of the secondary fragments were matched with the mzCloud network database and the local Chinese medicine component database OTCML respectively. Retain the compounds whose peak area threshold is greater than 80,000, the rst-and second-level mass deviation is less than 5 ppm, and the matching score is greater than 85, and then compare with the reference substance and related literature to identify the compound.

Results
Identi cation of components in the sample by HPLC -Q-Orbitrap HRM The ( Figure 2) and ( Figure 3) stands the total ion chromatogram of sample and mixed reference solution both in positive mode and negative mode respectively. Combined with the accurate relative molecular mass, fragmentation rules elucidation detected by the positive and negative ion mode, the molecular formula was calculated by the Compound Discoverer 3.0 software within the range of 5 ppm mass deviation, and match the available databases like mzCloud and the OTCML, at the same, referring to relevant literatures. Then, a total of 74 components, including 2 chromone, 23 avonoids, 10 alkaloids, 9 phenolic acids, 8 nucleosides and nucleobases, and 22 other componets, were identi ed from GSZG. The results of identi cation were show in Table 1.

Identi cation of chromone compounds
Chromone compounds are characteristic compounds of Saposhnikoviae Radix, and it is reported in the literature that these compounds have the effect of inhibiting arthritis [20]. Through research and analysis, it is concluded that this type of compound has a greater abundance in the positive ion mode under the conditions of the rst-level mass spectrum, mainly in the form of [M+H] + , by consulting related literature, it is found that the secondary mass spectrometry of this type of compound mainly loses neutral fragment ions such as glucoside (162) [21][22][23], the MS/MS spectrum is shown in (Figure 4), and MS 2 fragmentation pathway of compound 62 is shown in (Figure 5)

Identi cation of Flavonoids compounds
Flavonoids are a kind of obvious biologically active compounds widely distributed in Chinese medicinal herbs. According to literature reports, these have many pharmacological e cacy like neuroprotective, anti-in ammatory, anti-oxidant and anti-tumor effects [24][25][26][27].Such compounds are often combined with pentose, Hexose, and hexuronic acid, they are mainly divided into avonoids, avonols, dihydro avonoids, iso avones, chalcones, etc depending on the type of structure. Through research and analysis, it is concluded that this type of compounds is roughly similar in cleavage, except for iso avones, these compounds can be detected in both positive and negative ion modes under the condition of rst-order mass spectrometry, and the characteristic fragments of glycosides were mainly lost in the mass/mass spectrometry, such as pentose (132), Hexose (162), hexuronic acid (176), and then neutral molecular fragments such as CHO (15) [28,29] which provided evidence that compound 53 could be attributed to Liquiritin. Compound 67 obtained the quasi-molecular ion m/z 549.16272 [M-H]in the negative ion mode, and the predicted molecular formula was C 26 H 30 O 13 , which was similar to the ion fragments obtained by the cleavage of compound 53, they both had 417, 255, 153, 135, 119 and 91 ion fragments Searching database and comparing the literature [28,30] it is speculated that compound 67 was Liguiritigenin-7-O-β-D-apiosyl-4'-O-β-D-glucoside the MS 2 spectrum of compound 53 and 67 is shown in (Figure 6), and MS fragmentation pathway of compound 53 and 67 is shown in (Figure 7).

Identi cation of Alkaloid compounds
Alkaloid compounds are a kind of obvious biologically active ingredients widely distributed in Chinese medicinal materials, but most of these compounds have obvious side effects. According to literature reports, such compounds have signi cant anti-in ammatory and anti-tumor effects [31][32][33]. Through research and analysis, it is concluded that this type of compound has a greater abundance in the positive ion mode under the conditions of primary mass spectrometry, and mainly exists in the form of [M+H] + . Looking up related literature in the MS/MS spectrum, it is easy to lose the substituents, such as hydroxyl, amino, methoxy, acetyl, benzoyl, etc., thereby losing CO (28)

Identi cation of Nucleosides and nucleobases compounds
Nucleosides and nucleobases compounds are widely found in Chinese medicine herbs. Which led to ignored by researchers, however, these compounds have a wide range of biological activities. it has identi ed 8 from GSZG. The cleavage characteristic of this class of compounds is that losing one molecule of ribose, and the nucleobases was lost neutral molecular fragments such as NH 3 . Compound 22 obtained the molecular m/ z 268.1041 [M+H] + in the positive ion mode, and the molecular formula was predicted to be C 10 H 13 N 5 O 4 . In the secondary mass spectrometry, only m/z 136.06178 strong fragment ions existed, through database search and relevant literature [38] comparison, it was concluded that compound 22 was Adenosine. The secondary mass spectrum of the compound and the possible fragmentation pathway is exhibited (Figure 15) (Figure 16).

Other types of compounds
In addition, this study also identi ed sugars, amino acids, coumarins, vitamins, and terpenoids from GSZG, which found in a variety of Chinese herbal medicines. In this experiment, through database comparison, ve carbohydrate compounds were found. Under the negative ion mode, these compounds have the same fragment ions, such as m/z=59, m/z=71, m/ z=89, m/z=179. However, some compounds, such as compound 45, compared with the reference substance, and it was concluded that the compound was paeoni orin which only existed in Paeoniae Radix Alba, and the cleavage law mainly includes breaking glycosidic bonds, removing sugar, benzoic acid, etc. netutral molecules, forming characteristic fragment ions with m/z=121 and m/z=121 in the negative ion mode.

Discussion
UPLC-Q-Orbitrap HRMS has many advantages like high sensitivity, high scoregood resolution and quality stability [39], especially suitable for the screening, identi cation and rapid identi cation of the main chemical components in complex Chinese medicine prescriptions. GSZD was listed in the rst batch of classic recipes and was approved to be developed into granules [18]. According to relevant policy requirements, the members of this research group conducted related studies on the material standards of GSZD and the development of GSZG in the early stage, for the chemical constituents, using UPLC-Q-TOF-MS method to analysis the GSZD [14,40,41]. Traditional Chinese medicine prescriptions work synergistically with their many medicinal herbs. In the process of torment, different medicinal materials act synergistically to produce many new compounds. In this study, it was found that there were identi ed some new components which never reported, like Afzelin, Sakuranetin. Furthermore, it was identi ed many amino acids, carbohydrates under experimental conditions, which inferred the sample preparation technology in some extent. These studies will offer helpful information for clarifying bioactive components and preparation technology of GSZG.

Conclusion
In the present work, a strategy was proposed to pro le the chemical constituents found in GSZG with the help of UPLC-Q-Orbitrap HRMS. The nding revealed the fact that chemical constituents maybe the main ingredients existing in GSZG, followed by the provision of the unique data, the research provided more information of chemical components in GSZG in this eld. In addition, at present, the research group is developing the classic Chinese prescription GSZD into granules. Now, through the analysis of the chemical composition of GSZG produced in Japan, it will help the research group to prepare GSZG, as well as set the quality standard for the prepared GSZG.

Authors' contibutions
Qinwan Huang and Yongxiang Gao are both the corresponding authors on the study. Tingna Li and Hairong Zeng are rst authors and responsible for screening experiment, processing the data and writing the paper. Xiaorui Zhang, Yuanyuan Ren and Yijia Zeng helped in organizing the information and giving some suggestion. All authors read and approved the nal manuscript.

Funding
Availability of data and materials The research data generated from this study is included within the article.   Figure 1 The composition of GSZD Figure 4 The MS/MS spectrum of compound 62 Figure 5 The possible fragmentation pathway of compound 62 Figure 6 The MS2 spectrum of compound 53 and 67 The possible fragmentation pathways of compound 53 and 67 The possible fragmentation pathway of compound 24 The MS/MS spectrum of compound 24 Figure 10 The possible fragmentation pathway of compound 51 Figure 11 The MS/MS spectrum of compound 51 Figure 12 The MS/MS spectrum of compound 28

Figure 13
The MS/MS spectrum of compound 42 Figure 14 The possible fragmentation pathways of compound 28 and 42

Figure 15
The MS/MS spectrum of the compound 22

Figure 16
The possible fragmentation pathway compound 22