Applying Four-Step Characteristic Ion Filtering with HPLC-Q-Exactive MS/MS Spectrometer Approach for Rapid Compound Structures Characterization and Major Representative Components Quantification in Modified Tabusen-2 Decoction

Modified Tabusen-2 decoction (MTBD) is traditional Chinese Mongolia medicine, mainly used to treat osteoporosis. However, the precise material basis of this prescription is not yet fully elucidated. Herein, we establish an HPLC-Q-Exactive MS/MS spectrometer method with four-step characteristic ion filtering (FSCIF) strategy to quickly and effectively identify the structural features of MTBD and determine the representative compounds content. The FSCIF strategy included database establishment, characteristic ions summarization, neutral loss fragments screening, and secondary mass spectrum fragment matching four steps. By using this strategy, a total of 143 compounds were unambiguously or tentatively annotated, including 5 compounds which were first reported in MTBD. Nineteen representative components were simultaneously quantified with the HPLC-Q-Exactive MS/MS spectrometer, and it is suitable for eight batches of MTBD. Methodology analysis showed that the assay method had good repeatability, accuracy, and stability. The method established above was successfully applied to assess the quality of MTBD extracts. Collectively, our findings enhance our molecular understanding of the MTBD formulation and will allow us to control its quality in a better way. At the same time, this study can promote the development and utilization of ethnic medicine.


Introduction
Tabusen-2 decoction (TBD) is composed of Echinops latifolius Tausch (ELT) and Eucommia ulmoides Oliver (EU) [1]. On this basis, Modified Tabusen-2 decoction (MTBD) adds Panax notoginseng (PN) and Carthamus tinctorius L. (CT) [2]. Osteoporosis is a common orthopedic disease, especially in the elderly and postmenopausal women in China. TBD is a traditional classic prescription; it has been used to treat osteoporosis for centuries [3]. e literature shows that MTBD has the effect of treating osteoporosis; it can also be used to promote blood circulation, relieve swelling, relieve pain, continue muscles and bones, and treat soft tissue contusions, crush injuries, joint sprains, trauma, and open trauma caused by surgery [4,5]. e chemical compositions of each herb are various, having different pharmacological effects according to past reports. ELT, a traditional Chinese Mongolia herb, contained isochlorogenic acid A (ICGAA), chlorogenic acid, and other phenylpropanoids [6,7], which are the main active components in herb. e pharmacological mitigation of ELT on osteoporosis of postmenopausal women was also reported [8]. EU is enriched with lignans and iridoids, including geniposidic acid (GPA) and pinoresinol diglucoside (PDG), having obvious antihypertensive effect [9]. In recent years, EU has attracted considerable attention because of its antiosteoporosis, antisenile dementia, antiaging, anti-inflammatory, antithrombotic, and antitumor activities [10,11]. e flavonoids are the main active components of CT, with the efficacy of promoting blood circulation, removing blood stasis, and relieving pain [12]. Varieties of natural pigments isolated from CT, such as yellow pigments and red pigments [13], not only have pharmacological functions but also have some nutritive value. Furthermore, triterpenoid saponins are main active constituents in PN, which are widely used for promoting blood clotting, relieving swelling, and alleviating pain [14].
In accordance with traditional Chinese medicine (TCM), traditional Mongolia medicine (TMM) is characterized with multiple components and multiple targets and plays different roles in clinical therapy. is means that it is a great challenge to explain the main chemical composition of MTBD by traditional analytical methods. In particular, the presence of isomers makes its separation and analysis more difficult. In order to solve this problem, some researchers have used the methods of mass defect, relative mass defect, neutral loss filtering (NLF), mass defect filtering, and precursor ion to characterize the chemical structure in TCM or TMM prescription [15][16][17][18][19]. It has vital-important reference value for our following experiment. With the promotion of high-resolution mass spectrometry [20,21], we propose an FSCIF strategy for substructure recognition, which can significantly improve the detection effectiveness, accuracy, and sensibility. is analysis program shows obvious efficiency (reduce data processing time) and intelligence (simplify the process of structural identification).
Xie et al. [22] determined hydroxysafflor yellow A, notoginsenoside R 1 , ginsenoside Rg 1 , and ginsenoside Rb 1 with HPLC, but there are disadvantages of insufficient sensitivity and long running time (40 min). Hua et al. [23] established an HPLC-ELSD method to quantify the content of notoginsenoside R 1 , ginsenoside Rg 1 , and ginsenoside Re in PN but did not determine the content of the main components of ELT, EU, and CT. Hua et al. [24] conducted three different experiments by using HPLC, Ultraviolet detection, and ELSD methods and finally measured the content of representative components of ELT, EU, PN, and CT. But the shortcomings of this method are cumbersomeness and low responsiveness and they cannot be ignored. On the other hand, the previous literature has qualitatively analyzed the ingredients in a single medicinal material; it is not enough to explain the overall structure of MTBD due to the interaction between temperature and herbs in the process of decoction.

MTBD Sample and Standard Solutions Preparation.
Sample preparation was a critical step for precise and convincing detection by the HPLC-Q-Exactive MS/MS spectrometer method. e MTBD samples were prepared according to our previous extraction process, and the whole operation process was in line with the basic operation safety regulations of the laboratory. EU, ELT, and CT herbal materials were powdered and sieved through 40 meshes for later extraction. A total 3.6 g of MTBD powders was accurately weighed (including 1.6 g of EU, 1.2 g of ELT, and 0.8 g of CT) and placed in a 250 mL round-bottomed flask. ese powders were immersed in 50 mL ethanol: water (6 : 4, V/V) mixture and weighed and then reflux extracted twice, 90 min for each reflux. Taking into account the recovery rate of PN powder, 0.4 g PN was added before the last extraction. After merging and mixing, the solution was filtered through a 0. 45   Evidence-Based Complementary and Alternative Medicine 3 digoxin was selected as the internal standard. ese standard solutions were diluted with mobile phase to final concentration (Table S1) before injection into HPLC-Q-Exactive MS/MS spectrometer.

Chromatography and Mass Spectrometry Conditions.
e characterization and quantification of MTBD sample extracts were analyzed using a ermo HPLC-Q-Exactive MS/MS spectrometer system (HPLC, UltiMate 3000, mass system, Quadrupole Exactive Orbitrap TM ). e qualitative analytical conditions were as follows: HPLC column, COSMOSIL C18 (250 mm e qualitative and quantitative mass parameters conditions were set up as follows: auxiliary gas heater temperature, 150°C; capillary temperature, 350°C; spray voltage, 3.5 kv; S-lens RF level, 50; sheath gas flow rate, 40 L; and auxiliary gas flow rate, 2 PSI. AGC was 3 × 10 6 in MS scan and 1 × 10 5 in MS/MS scan; IT was 100 ms in MS scan and 50 ms in MS/MS scan; resolution was 70000 in MS scan and 17500 in MS/MS scan; NCE was set as 30 v. Scanning range was 100-1500 m/z. Mass spectrometry uses full scan mode for analysis in positive ion mode and negative ion mode.

Method Validation.
e dependent variable was the ratio of the peak area of each analyte to the peak area of the internal standard, while the independent variable was set as the concentration value of each analyte; the least square regression was used to construct the standard curve equation.
e intraday and interday precisions and accuracies were assessed by analyzing each concentration level (low, medium, and high) of six repeated QC samples on the same day and three consecutive days, respectively. Sample stability was investigated after the extracts were kept at room temperature for 0 h, 6 h, 12 h, and 24 h. Add the mixed control solution equal to the content of each analyte in the sample to the MTBD sample, repeat the preparation of 6 solutions, and calculate the recovery according to the following formula: (1)

Results and Discussion
3.1. Construction of the Identification Strategy. Each type of compounds has its similar core and skeleton. On this basis, the characteristic ion will be produced, which provides us with new ideas for identifying these structures. In addition, FSCIF is especially suitable for compounds with the same structural type containing similar fragmentation pathways with some characteristic ions. Correspondingly, an FSCIFbased and substructure scanning strategy will be used for rapid identification of MTBD structures. e analytical strategy is shown in Figure 2. e compounds in MTBD were characterized by HPLC-Q-Exactive MS/MS spectrometer method with FSCIF strategy, including the following steps: (1) established the self-building chemical database of MTBD according to literature and online database; (2) comprehensively summarized characteristic ions for each compound type to conduct global identification of the ingredients in MTBD; (3) rapidly screened relevant structure information by neutral loss fragments (NLF) to conform the sugar type, conjunction position, and other information; (4) concluded the precise compound structure through high-precision MS/MS data. e typical total ion chromatograms (TICs) of MTBD by HPLC-Q-Exactive MS/MS spectrometer system in positive and negative ion modes are shown in Figure 3. 143 compounds were annotated through high-precision MS/MS data, including 51 triterpenoid saponins, 28 flavonoids, 20 phenylpropanoids, 15 iridoids, 12 lignans, 11 polyphenols, and 6 other types (Table 1), in which 5 compounds were first reported in MTBD and 20 compounds were unambiguously identified by comparison with reference standards. ese 143 components' structures are shown in Figure S1.              Figure S2 Additionally, the sugar type and its junction position were concluded with the application of NLF strategy. e position of sugar fragments on the aglycon was relatively fixed (C3, C6, and C12), and the main types of sugars were glc (162.02 Da), rha (146.01 Da), and xyl (132.02 Da); the linkage between sugars is mainly l-2 and 1-6. In this experiment, nineteen compounds (62, 82, 83, 84, 89, 90,  91, 92, 93, 94, 98, 99, 102, 105, 106, 120, 124, 125, and 133

3.2.3.
Identification of Phenylpropanoids. Phenylpropanoids and their derivatives, including monocaffeoylquinic acids, biscaffeoylquinic acids, and caffeoylquinic acid derivatives, were main components widely present in MTBD. Some papers [28] have previously shown that phenylpropanoids have multifaceted effects which include anti-inflammatory, antioxidant, antimicrobial, and antidiabetic activities and exhibit renoprotective, hepatoprotective, and cardioprotective effects. By using the FSCIF strategy, twenty phenylpropanoids were found; the ions at m/z 191.05 Da and 179.03 Da represented the base peaks of quinic acid, whereas ions at m/z 161.02 Da and 135.04 Da represented the base peaks of caffeic acid. Chlorogenic acid is an ester of caffeic acid and quinic acid, which indicates that chlorogenic acid contains the feature ions of both caffeic acid and quinic acid. A total of twelve compounds (4, 14, 22, 29, 30, 38, 45,

Identification of Iridoids.
e most basic core of iridoids is iridoid alcohol, containing cyclic ethers and alcoholic hydroxyl groups, which imply that the basic skeleton of iridoid glycosides contains a characteristic dihydropyran ring which is cis-connected to a cyclopentane unit structure. A total of 15 iridoids were detected [M − H] − in negative ion mode. In the ESI − mode, the fragment ion 2,7 F0 − ion at m/z 101.02 was obtained by the fragmentation of the aglycon part of the excimer ion, which was a characteristic ion to annotate the structure of the excimer ion [29,30]. According to the literature [11], the ion at m/z 147.03 was the prominent ion of iridoids. Compounds 5,8,11,13,  were characteristic fragments of compound 7, which was tentatively annotated as geniposidic acid. To sum up, the iridoids were easier to lose the glucose neutral fragment ion 162.02 Da and obtain aglycon fragment ions and then the aglycon ions decarboxylated or dehydrated to become a series of fragments.

Identification of Lignans.
A large number of the bisepoxylignans and monoepoxylignans combine with glucose to form monoglycoside or diglycoside. erefore, the majority of them could lose glycosyl and methyl neutral fragments first and then lose one or two molecular of CH 2 (3, 6, 9, 17, 18, 20, 23, 24, 25, 34, and 35) were recognized by FSNLF analysis. Because of the presence of hydroxyl and carboxyl groups, these compounds were filtered by 18.01 Da (H 2 O) and 44.01 Da (CO 2 ). In addition, six other compounds were identified by comparison with the literature.

Quantification of 19 Major Compounds in MTBD.
e 19 compounds quantified were the screening of osteoporosis targets by network pharmacology in the early stage of our laboratory, and then the representative and top ranked compounds were selected. Methodology analysis showed that the assay method of 19 compounds (including three pairs of isomers) had good repeatability and stability.

Specificity.
e extracted ion chromatograms (EICs) of blank sample, standard mixture sample, and MTBD extracts sample are presented in Figure 4. Nineteen compounds in MTBD extracts were separated within 25 minutes, where baseline separation of each compound was achieved and no obvious signal noises occurred around determinate peak. Additionally, no interferences were detected between the three isomers.

Linearity and Lower Limit of Quantification.
ree batches of standard curve solutions with six different concentrations were prepared. e typical standard curves were assessed by using DAS 2.0 software with the quadratic weight (W � 1/C 2 ). e dependent variable was the ratio of the peak area of each analyte to the peak area of the internal standard, while the independent variable was set as the concentration value of each analyte; the least square regression was used to construct the standard curve equation. e standard curves and correlation coefficients are listed in Table 2, proving the calibration curves of the components with a good linearity over the studied concentration range. e lower limit of quantification (LLOQ) for each analyte was all with signal-to-noise ratio higher than 10, which was sufficient to perform quantitative studies of MTBD extracts.

Precision and Accuracy.
ree batches of quality control samples were prepared according to three concentration levels. Each concentration was analyzed with 6 duplications. e intraday precision values were between 1.13% and 6.66%, and the interday ones were between 2.42% and 10.62% and accuracy ranged from 86.11% to 114.27%. e above results demonstrated the acceptable precision and accuracy of the present method.

Repeatability and Stability.
Six MTBD sample extracts were prepared on the same day according to Section 2.2. e repeatability of 19 components was within 6.26% relative standard deviation (RSD).
Sample stability was investigated after the extracts were kept at room temperature for 0 h, 6 h, 12 h, and 24 h. e stability results of 19 compounds are summarized in Table 3; the acceptability of the data was within 3.92% deviation from the 0 h sample values, which indicated that a large number of samples could be stable in each analytical run.

Application to Samples Modified Tabusen-2 Decoction (MTBD).
e method established above was successfully utilized for quantitative studies of MTBD extracts, as shown in Table S2. Eight batches of MTBD samples prepared with different herb sources were determined by using the above mature method. e herb formulation of each batch is listed in Table S3.
ere is an indication of the fact that the concentrations of 19 compounds varied significantly in MTBD extracts; the content of flavonoids was the highest, followed by saponins ( Figure S4), which attracted the attention of herb quality in picking as well as in circulating during the market. It can be seen from the quantitative research results of different batches of MTBD that we need to strictly control the quality of herb because this is the guarantee of their clinical efficacy and safety.

Discussion
Although the isolation and purification before biological activity evaluation are a traditional strategy of exploring material basis in TMM, the time-consuming and labor-intensive characteristics cannot be neglected. In quantitative experiments, Ultraviolet (UV) detector is exceedingly common for flavonoids, phenylpropanoids, and other UV-absorbing compounds [32], while it is not applicable to the analysis of saponin. Although the detection of saponin could be enabled by evaporative light scattering detector (ELSD), the sensitivity during the test procedure should also be taken into account [33]. Herein, in order to shorten the analysis time, improve the analysis sensitivity, and simultaneously determine UV-absorbing compounds and non-UV-absorbing compounds, high performance liquid chromatography coupled with mass spectrometry (HPLC-Q-Exactive MS/MS spectrometer) approach [34], as a high efficiency, is employed in this study to separate and identify the material basis in MTBD. Additionally, the existence of isomer (ICGAA with 1,5-DQA, GE with APG, LT with KPF) in MTBD increases the difficulty of separation and analysis [35]. e chromatographic conditions in quantitative analysis need to be optimized carefully during the present research.
In order to achieve better separation effect for three pairs of isomers in MTBD, the mobile phase was screened in this experiment. e peak of each component was with It was found through analysis that the contents of the 19 components differ in MTBD prepared from different batches of crude drugs; this might be because the crude drugs of different batches were different in origin, growing environments, and harvest time.
is has aroused our attention in all aspects of picking and transportation. e presence of moisture will affect the determination of the content of the active ingredients in the medicinal materials. erefore, the near-infrared method was used in the study to detect the moisture content in the relevant medicinal materials to ensure the final quantitative accuracy of the effective ingredients [36]. Refluxing was used to prepare the MTBD in the present study [37]. Furthermore, some literatures [38][39][40][41][42] have carried out assays on HSYA, RU, QC, G-Rb 1 , G-RG 1 , NG-R 1 , G-Re, FA, LT, KPF, APG, and GE; but the HPLC-Q-Exactive MS/MS spectrometer approach displayed distinct superiority with desirable resolution and Lower LLOQ. e previous literature [43,44] measured the content of CA, 1.5-DQA, GPA, PDG, and CGA, but it took too long (60 minutes) and restricted its modern development. Some studies [45,46] have shown the contents of ICGGA and A-7-O-G; on this basis, we can have a wider linear range and have greater reference value for the formulation of the content of different batches of samples.

Conclusions
Based on HPLC-Q-Exactive MS/MS spectrometer with FSCIF approach to rapid detection of structure fragment and quantification of major representative components in MTBD, 143 compounds with seven chemical categories were unambiguously or tentatively identified. is study not only enriched the cleavage law of MTBD compounds but also established an approach for the accurate search and discovery of active components from complex mixtures. e repeatability, accuracy, stability, linearity, recoveries, and reproducibility of quantitative analysis all meet the criteria for acceptability of quantitative studies.  e determination of 19 compounds in MTBD extracts in different batches was obtained to monitor the quality of each prescription, which facilitates the better development of quality evaluation technique in MTBD and will help for further exploration of quality control of MTBD. e 19 compounds determined based on the qualitative and quantitative results are the major components of the MTDB. is experiment can provide a research foundation for subsequent pharmacokinetic studies and formulation of quality standards.
All in all, we compared the differences in the content of the same compound in the same herbs. Our quantitative method can determine 19 compounds in a short time (25 minutes), with a wider linear range and lower LLOQ. On the other hand, we compared the content difference of the same compound in different herbs, and the content fluctuation range is relatively large, which may be related to the processing, compatibility, and the changes in the decocting process of herbs. e content range of the 19 compounds that we have measured can provide the fluctuation range of the compound content when formulating quality standards in the future and help formulate content determination standards for preparations.
is qualitative and quantitative analysis of MTBD could provide a new tool for the quality control of this preparation or its related TCM.
Data Availability e methodological data and structural data used to support the findings of this study are included within the article. e cleavage pathways data used to support the findings of this study are included within the Supplementary Materials.