Analysis method for chemical components of salvia miltiorrhiza and pseudo-ginseng drug pair based on offline two-dimensional liquid chromatography and mass spectrometry

Abstract

The invention discloses an off-line two-dimensional liquid chromatography and mass spectrometry-based chemical component analysis method for a salvia miltiorrhiza-panax notoginseng drug pair, which comprises the following steps: step (1), preparing a reference substance into a mixed reference substance solution by using methanol as a solvent; (2) preparing a one-dimensional chromatographic test solution from the salvia miltiorrhiza and pseudo-ginseng drug pair by adopting an ultrasonic extraction method; (3) carrying out preparative separation on the one-dimensional chromatography test solution by adopting first-dimensional chromatography, and collecting fractions in sections according to peak appearance conditions; the collected fractions are blow-dried, redissolved and centrifuged, and then supernate is taken; and (4) respectively separating the mixed reference substance solution and the two-dimensional chromatography test solution on a second-dimensional chromatography, carrying out mass spectrometry detection, and carrying out comparative analysis on the detection results to realize qualitative analysis of the chemical components in the salvia miltiorrhiza and pseudo-ginseng drug pair. The method can solve the problem of incomplete characterization of the chemical components of the salvia miltiorrhiza and pseudo-ginseng drug pair caused by limited separation capacity of the existing chromatographic separation method on the chemical components in the salvia miltiorrhiza and pseudo-ginseng drug pair.

Description

Technical Field

[0001] This invention relates to the field of component analysis technology for Tanshinone and Panax notoginseng pairs. Specifically, it is a method for chemical component analysis of Tanshinone and Panax notoginseng pairs based on offline two-dimensional liquid chromatography coupled with mass spectrometry. Background Technology

[0002] The Danshen-Sanqi herbal pair is one of the most frequently used blood-activating and stasis-removing herbal pairs in clinical practice. Danshen and Sanqi are rich in chemical components, mainly including phenolic acids (such as salvianolic acid B, tanshinone, rosmarinic acid, caffeic acid, etc.), diterpenoid quinones (tanshinone IIA, cryptotanshinone, tanshinone I, etc.), saponins (Sanqi saponin R1, ginsenoside Rg1, ginsenoside Re, etc.), polysaccharides, lactones, flavonoids, and amino acids. Among these, the phenolic acids and diterpenoid quinones in Danshen, and the saponins in Sanqi, are the main active ingredients in the herbal pair. Currently, most studies on the Danshen-Sanqi herbal pair focus on its pharmacological effects and mechanisms; there are no reports of a comprehensive qualitative analysis of the chemical components of Danshen and Sanqi. Chemical components are the material basis for the formulation of traditional Chinese medicine, and clarifying the material basis of traditional Chinese medicine is an important way to reveal the efficacy mechanism of traditional Chinese medicine and improve its therapeutic effect. Therefore, there is an urgent need to utilize modern scientific and technological methods to comprehensively characterize the chemical components of the *Salvia miltiorrhiza* and *Panax notoginseng* herb pairs, elucidate their material basis, and thus provide data support for their quality control. In recent years, UPLC-Q-Orbitrap HRMS (ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry) has been widely applied to the analysis of complex samples due to its high efficiency, high sensitivity, and high resolution. However, because the composition of *Salvia miltiorrhiza* and *Panax notoginseng* herb pairs is relatively complex, conventional one-dimensional liquid chromatography separation methods have low peak capacities and relatively limited separation capabilities, resulting in the inability to achieve good separation of some compounds. This may lead to a lack of comprehensiveness in the study of the chemical components of *Salvia miltiorrhiza* and *Panax notoginseng* herb pairs. Therefore, it is necessary to find better analytical methods for the chemical components of *Salvia miltiorrhiza* and *Panax notoginseng* herb pairs to achieve a more in-depth and comprehensive characterization of their chemical composition. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention is to provide a method for the analysis of chemical components of Panax notoginseng pairs based on offline two-dimensional liquid chromatography coupled with mass spectrometry, so as to solve the problem of incomplete characterization of the chemical components of Panax notoginseng pairs caused by the limited separation ability of existing chromatographic separation methods.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0005] A method for chemical component analysis of *Salvia miltiorrhiza* and *Panax notoginseng* based on offline two-dimensional liquid chromatography coupled with mass spectrometry includes the following steps:

[0006] Step (1) Preparation of mixed reference solution: Using methanol as solvent, the reference standard is prepared into a mixed reference solution; Step (2) Preparation of one-dimensional chromatographic test solution: The one-dimensional chromatographic test solution is prepared by ultrasonic extraction of the Danshen-Sanqi herb pair; Step (3) Preparation of two-dimensional chromatographic test solution: Hydrophilic interaction liquid chromatography is used as the first dimension of chromatography. The one-dimensional chromatographic test solution is prepared and separated on a semi-preparative liquid chromatography system. The fractions are collected in segments according to the peak conditions. The collected fractions are dried by nitrogen gas and then reconstituted with ethanol aqueous solution. Finally, the reconstituted liquid is centrifuged and the supernatant is taken to prepare the two-dimensional chromatographic test solution; Step (4) The mixed reference solution and the two-dimensional chromatographic test solution obtained in step (3) are separated and detected by mass spectrometry on an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry system using reversed phase chromatography as the second dimension of chromatography. The detection results are compared and analyzed to achieve qualitative analysis of chemical components in the Danshen-Sanqi herb pair.

[0007] The above-mentioned method for chemical component analysis of *Salvia miltiorrhiza* and *Panax notoginseng* based on offline two-dimensional liquid chromatography-mass spectrometry, in step (1), the mass concentration of quercetin in the mixed reference solution is 70-80 μg / mL, the mass concentration of notoginsenoside R1 is 180-200 μg / mL, the mass concentration of ginsenoside Rb1 is 100-110 μg / mL, the mass concentration of ginsenoside Rb2 is 90-100 μg / mL, the mass concentration of ginsenoside Rc is 130-140 μg / mL, the mass concentration of ginsenoside Rd is 160-170 μg / mL, the mass concentration of ginsenoside Rg1 is 90-100 μg / mL, and the mass concentration of 20(S)-ginsenoside Rg3 is 90-100 μg / mL. The concentrations of the following ingredients were measured: ginsenoside Re (80-90 μg / mL), salvianolic acid B (135-145 μg / mL), salvianolic acid C (215-225 μg / mL), dihydrotanshinone I (65-75 μg / mL), salvianolic acid A (80-90 μg / mL), tanshinone IIA (60-70 μg / mL), tanshinone neoketone (70-80 μg / mL), tanshinone I (70-80 μg / mL), cryptotanshinone (170-180 μg / mL), sodium tanshinone (180-190 μg / mL), and rosmarinic acid (90-100 μg / mL).

[0008] In the above-mentioned method for chemical component analysis of the Danshen-Sanqi herb pair based on offline two-dimensional liquid chromatography coupled with mass spectrometry, in step (2), the solvent used for ultrasonic extraction is a methanol aqueous solution or an ethanol aqueous solution, the volume fraction of methanol in the methanol aqueous solution is 50-80%, and the volume fraction of ethanol in the ethanol aqueous solution is 50-80%; during ultrasonic extraction, the ultrasonic treatment power is 100-200W, the ultrasonic frequency is 30-50kHz, and the ultrasonic treatment time is 20-40min; the mass ratio of Sanqi powder to Danshen powder in the Danshen-Sanqi herb pair is 1:(0.5-2); the mass-volume ratio of the Danshen-Sanqi herb pair to the solvent is 0.03-0.05g / mL; both Danshen powder and Sanqi powder pass through a 65-mesh sieve.

[0009] In the above-mentioned method for chemical composition analysis of Danshen and Panax notoginseng drug pair based on offline two-dimensional liquid chromatography and mass spectrometry, in step (3), when each fraction is dried with nitrogen, the temperature of the drying nitrogen is 50-70℃; the volume fraction of ethanol in the ethanol aqueous solution used for redissolution is 60-80%; the centrifugation rate is 14000-15000 rpm and the centrifugation time is 10-15 min.

[0010] The technical solution of the present invention achieves the following beneficial technical effects:

[0011] This invention presents a method for the chemical composition analysis of *Salvia miltiorrhiza* and *Panax notoginseng* herb pairs using offline two-dimensional liquid chromatography-mass spectrometry (HPLC-MS). The offline 2D-LC-Q-Orbitrap HRMS system significantly improves peak capacity and better separates chemical components compared to traditional one-dimensional liquid chromatography. Using this method, chemical components in a 1:1 ratio of *Salvia miltiorrhiza* and *Panax notoginseng* herb pairs were separated and characterized, ultimately identifying 139 compounds, including 59 saponins, 25 phenylpropanoids, 25 terpenoids, 7 flavonoids, and 23 other compounds. Among these, 25 new components were identified compared to the results of UPLC-Q-Orbitrap HRMS, further enriching the chemical composition database of *Salvia miltiorrhiza* and *Panax notoginseng* herb pairs. This method can provide preliminary analysis of the material basis of *Salvia miltiorrhiza* and *Panax notoginseng* herb pairs, offering data support for quality control of these pairs. It also provides insights and research strategies for the study of the material basis of herb pairs and other complex samples. Attached Figure Description

[0012] Figure 1A and Figure 1B The total ion chromatograms of the Danshen-Sanqi herb pair samples under positive and negative modes were analyzed by UPLC-Q-Orbitrap HRMS in the examples.

[0013] Figure 2A and Figure 2BThe total ion chromatograms of the mixed reference solution sample under positive and negative modes were analyzed by UPLC-Q-Orbitrap HRMS in the examples.

[0014] Figure 3A and Figure 3B The total ion chromatograms of the Danshen samples under positive and negative modes were analyzed using UPLC-Q-Orbitrap HRMS in the examples, respectively.

[0015] Figure 4A and Figure 4B The images show the total ion chromatograms of Panax notoginseng samples under positive and negative modes, respectively, based on the UPLC-Q-Orbitrap HRMS method in this embodiment of the invention.

[0016] Figure 5A , Figure 5B , Figure 5C and Figure 5D The following are chromatograms of mixed reference standards used in the embodiments of the present invention based on the orthogonality study of offline two-dimensional liquid chromatography and mass spectrometry: the mobile phase is methanol-0.1% formic acid water, the 1D column is GOWON Amide (A), and the 1D column is Xbrigde Amide (B).

[0017] Figure 6A and Figure 6B The images are chromatograms of test samples with different mobile phase compositions based on offline two-dimensional liquid chromatography and mass spectrometry in the embodiments of the present invention at detection wavelengths of 203 nm and 270 nm, respectively.

[0018] Figure 7A and Figure 7B The images show the chromatograms of the test solution at different column temperatures and detection wavelengths of 203 nm and 270 nm, respectively, in the embodiments of the present invention based on offline two-dimensional liquid chromatography and mass spectrometry.

[0019] Figure 8A and Figure 8B The images show the chromatograms of the test sample at different flow rates and detection wavelengths of 203 nm and 270 nm, respectively, in the embodiments of the present invention based on offline two-dimensional liquid chromatography and mass spectrometry.

[0020] Figure 9 In this embodiment of the invention, chromatograms of 2D fractionated test solutions are prepared based on offline two-dimensional liquid chromatography and mass spectrometry.

[0021] Figure 10 Offline two-dimensional liquid phase system orthogonality evaluation and analysis diagram in this embodiment of the invention;

[0022] Figure 11 A bar chart of the orthogonality evaluation results of the offline two-dimensional liquid phase system in this embodiment of the invention;

[0023] Figure 12A, Figure 12B , Figure 12C , Figure 12D , Figure 12E , Figure 12F , Figure 12G , Figure 12H , Figure 12I and Figure 12J The images show the total ion chromatograms of the 2D segmented test solution (Fr.1-Fr.10) in positive ion mode based on offline two-dimensional liquid chromatography and mass spectrometry in the embodiments of the present invention.

[0024] Figure 13 A flowchart of the offline two-dimensional liquid chromatography workflow in this embodiment of the invention. Detailed Implementation

[0025] I. Chemical Constituent Analysis of Tanshinone and Panax notoginseng Pairs Based on UPLC-Q-Orbitrap HRMS

[0026] 1. Experimental Materials

[0027] The danshen (Salvia miltiorrhiza) slices (batch number: 230902861) and sanqi (Panax notoginseng) slices (batch number: 230600321) were both purchased from Kangmei Pharmaceutical Co., Ltd. Associate Professor Long Fei of the School of Pharmacy, Chengdu University of Traditional Chinese Medicine, identified them as the dried roots and rhizomes of *Salvia miltiorrhiza* Bge. (Lamiaceae) and *Panax notoginseng* (Burk.) FHChen (Araliaceae), respectively, processed products according to the 2020 edition of the *Chinese Pharmacopoeia*. Danshen and Sanqi samples: Appropriate amounts of danshen and sanqi slices were pulverized and passed through a 65-mesh sieve. Danshen and Sanqi herb pair sample: Equal amounts of danshen and sanqi slices were pulverized, passed through a 65-mesh sieve, and mixed thoroughly.

[0028] The main instruments used in the experiment are as follows: Ultra-high performance liquid chromatography-quadrupole electrostatic field orbital trap high-resolution mass spectrometer (model Oribitrap 120, Thermo Fisher Scientific (China) Co., Ltd.); Ultra-high performance liquid chromatograph (model Vanquish core, Thermo Fisher Scientific (China) Co., Ltd.); Electronic balance (accuracy: 0.01%, model SQP, Sartorius Scientific Instruments (Beijing) Co., Ltd.); Electronic balance (accuracy: 0.01%, model SQP, Sartorius Scientific Instruments (Beijing) Co., Ltd.); CNC ultrasonic cleaner (model KQ-400DE, Kunshan Ultrasonic Instruments Co., Ltd.); Circulating water vacuum pump (model SHZ-Ⅲ, Shanghai Yarong Biochemical Instrument Factory); Electronic thermostatic heating mantle (model DZTW, Beijing Yongguangming Medical Instruments Co., Ltd.); High-speed traditional Chinese medicine pulverizer (model HS-500A, Xi'an Hardware and Medicine Factory, Yongkang City, Zhejiang Province); Ultrapure water system (model Milli-Q; Merck Chemical Technology (Shanghai) Co., Ltd.).

[0029] The reference standards and reagents used in the experiment are as follows: quercetin (batch number: HR20119B1), notoginsenoside R1 (batch number: HP4158B2), ginsenoside Rb1 (batch number: HR191017B3), ginsenoside Rb2 (batch number: HR1513W8), ginsenoside Rc (batch number: HR214W13), and ginsenoside Rd (batch number: HR2326W9). The purity of ginsenoside Rg1 (batch number: HR22809B1), 20(S)-ginsenoside Rg3 (batch number: HR1855W13), ginsenoside Re (batch number: HR2185B1), salvianolic acid B (batch number: HS19910B2), salvianolic acid C (batch number: HR4041W18), and dihydrotanshinone I (batch number: HS91225B3) was 98%. All of the following are produced by Baoji Chenguang Biotechnology Co., Ltd.: Tanshinone A (purity greater than 98.00%, batch number: PS020493), Tanshinone IIA (purity 99.71%, batch number: PS12090601), and Tanshinone Neoketone (purity greater than 98.00%, batch number: PS020627) are produced by Chengdu Pusi Biotechnology Co., Ltd.: Tanshinone I (purity 99.09%, batch number: MUST-18032207), Cryptotanshinone (purity 98.91%, batch number: MUST-18041115), Sodium Tanshinone (purity 99.87%, batch number: MUST-19052206), and Rosmarinic acid (purity 99.02%, batch number: MUST-18053110) are produced by Chengdu Mansite Biotechnology Co., Ltd.

[0030] Formic acid (chromatographic grade), formic acid (mass spectrometry grade), and acetic acid (chromatographic grade) were all produced by Shanghai Aladdin Biochemical Technology Co., Ltd.; acetonitrile (chromatographic grade) and acetonitrile (mass spectrometry grade) were both produced by Thermo Fisher Scientific (China) Co., Ltd.; and methanol (chromatographic grade) and ethanol (chromatographic grade) were both produced by Saen Chemical Technology (Shanghai) Co., Ltd.

[0031] 2. Methods and Results

[0032] 2.1 UPLC Chromatographic Conditions

[0033] Chromatographic column: Hypersil C18 (2.1 mm × 100 mm, 1.9 μm), with acetonitrile as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B. Preparation method of 0.1% formic acid aqueous solution: take 1 mL of formic acid and dilute to 1 L with water (all subsequent 0.1% formic acid aqueous solutions were prepared using this method); elute according to the gradient in Table 1; column temperature: 30℃; detection wavelengths: 203 nm and 270 nm; flow rate: 0.3 mL / min; injection volume: 3 μL.

[0034] Table 1 Gradient elution program

[0035]

[0036] 2.2 Mass Spectrometry Conditions

[0037] Electrospray ionization (ESI) was employed, with simultaneous detection in both positive and negative ion modes. The spray voltage was 3.4 kV in positive ion mode and -2.5 kV in negative ion mode. The ion source temperature was 350 °C, the sheath gas flow rate was 35 arb, the auxiliary gas flow rate was 10 arb, and the capillary temperature was 320 °C. Full MS / dd MS was used. 2 The scanning mode has a scanning range of m / z 100–1500; the full scan resolution is 60,000 FWHM, and the secondary scan resolution is 15,000 FWHM; high-energy collision-induced dissociation is performed at normalized collision energies of 20 / 40 / 60V.

[0038] 2.3 Preparation method of test solution

[0039] The test solution for the Danshen-Sanqi herb pair: Take about 0.4g of the Danshen-Sanqi herb pair (the ratio of Danshen-Sanqi herb pair is 1:1), accurately weigh it, place it in a stoppered conical flask, accurately add 10mL of 70% ethanol solution, stopper tightly, weigh it, sonicate (power 150W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% ethanol solution, shake well, filter it, and take the filtrate to obtain the test solution.

[0040] Salvia miltiorrhiza test solution: Take about 0.2g of Salvia miltiorrhiza sample, weigh accurately, and prepare it according to the above method.

[0041] Panax notoginseng test solution: Take about 0.2g of Panax notoginseng sample, weigh accurately, and prepare it according to the above method.

[0042] 2.3.1 Investigation of extraction solvent

[0043] The effect of different extraction solvents (water, 30% methanol, 30% ethanol, 50% methanol, 50% ethanol, 70% methanol, 70% ethanol, methanol, ethanol; here, the percentage concentration refers to the volume concentration of methanol or ethanol relative to water) on the extraction efficiency of *Salvia miltiorrhiza* and *Panax notoginseng* was investigated using ultrasonic treatment. The total number and area of ​​peaks at 203 nm and 270 nm were recorded, as shown in Table 2. The results showed that the separation effect was better when the extraction solvent was 70% ethanol, with the highest number of peaks and the largest total peak area. Therefore, 70% ethanol was ultimately selected as the extraction solvent.

[0044] Table 2 Results of the investigation of different extraction solvents

[0045]

[0046] 2.3.2 Examination of extraction time

[0047] Following the above method, using 70% ethanol as the extraction solvent, the effects of different extraction times (10 min, 15 min, 20 min, 30 min, 40 min, 60 min, and 90 min) on the extraction efficiency of *Salvia miltiorrhiza* and *Panax notoginseng* were investigated. The results are shown in Table 3. The highest number of total peaks was obtained when the ultrasonic extraction time was 30 min, while the largest total peak area was obtained when the ultrasonic extraction time was 40 min, but the difference in the number and area of ​​total peaks between the two was not significant. Ultrasonic extraction for 30 min yielded a relatively large number of peaks and a large peak area in a relatively short time; therefore, 30 min was determined as the optimal extraction time.

[0048] Table 3 Results of the investigation at different extraction times

[0049]

[0050]

[0051] 2.4 Preparation of Mixed Reference Solution

[0052] Accurately weigh appropriate amounts of 19 reference standards, including quercetin, and add methanol to prepare a solution containing, per 1 mL: quercetin 72 μg, notoginsenoside R1 190 μg, ginsenoside Rb1 106 μg, ginsenoside Rb2 96 μg, ginsenoside Rc 132 μg, ginsenoside Rd 166 μg, ginsenoside Rg1 94 μg, 20(S)-ginsenoside Rg3 93 μg, ginsenoside Re 87 μg, salvianolic acid B 141 μg, salvianolic acid C 219 μg, dihydrotanshinone I 70 μg, salvianolic acid A 87 μg, and tanshinone IIA. A mixed solution of 63 μg of tanshinone, 71 μg of tanshinone I, 75 μg of cryptotanshinone, 171 μg of tanshinone sodium, 185 μg of tanshinone, and 95 μg of rosmarinic acid was prepared and stored at 4°C.

[0053] 2.5 Sample Determination

[0054] Take the test solutions of Danshen and Sanqi, Danshen test solution, Sanqi test solution, and mixed reference solution, and perform the tests according to the above conditions.

[0055] 2.6 Data Processing

[0056] Data acquired by high-resolution mass spectrometry (HMS) was analyzed using Compound Discoverer 3.0 software to establish a workflow for identifying unknown compounds. After preprocessing including peak extraction, alignment, and noise reduction, the possible molecular formulas were deduced based on the precise molecular weight provided by the primary mass spectrometer. Secondary fragment information was compared with compound data in the mzCloud and mzVault databases, and compounds with a matching degree higher than 80% were screened for preliminary identification. Simultaneously, by comparing the structures and characteristic fragment ions of compounds contained in *Salvia miltiorrhiza* and *Panax notoginseng* with reported literature, a chemical composition mass spectrometry database was constructed. The theoretical quasi-molecular ion mass-to-charge ratio (m / z) of the compounds was calculated using a molecular weight calculator (6.6.0). Peak extraction and peak matching were performed on the acquired MS data using Xcalibur 3.0, and the results were compared with those from online databases, the self-built database, and reference standards to achieve accurate identification of the compounds.

[0057] 2.7 Composition Identification

[0058] The chemical composition of single-herb solutions of Salvia miltiorrhiza and Panax notoginseng, as well as their paired herb solutions, extracted with 70% ethanol solution by ultrasound was analyzed using UPLC-Q-Orbitrap HRMS technology. The total ion chromatograms of the Salvia miltiorrhiza and Panax notoginseng paired samples, mixed control, single-herb Salvia miltiorrhiza sample, and single-herb Panax notoginseng sample under positive and negative ion modes are shown below. Figures 1A to 1B , Figures 2A to 2B , Figures 3A to 3B and Figures 4A to 4B As shown. Following the data processing methods described above, and combining relevant literature with reference standards, a total of 119 compounds were identified from the 70% ethanol ultrasonically extracted Salvia miltiorrhiza and Panax notoginseng sample solution. These included 54 saponins, 20 phenylpropanoids, 22 terpenoids, 7 flavonoids, and 16 other compounds. The retention times and mass spectrometry data of 19 compounds were compared with the reference standards, leading to their definitive identification. Among these, 50 compounds originated from Salvia miltiorrhiza, mainly phenylpropanoids and terpenoids; 61 compounds originated from Panax notoginseng, mainly saponins; and 8 compounds were shared by both, mainly organic acids.

[0059] Based on the above results, saponins, phenylpropanoids, and terpenoids are considered to be the main components of the *Salvia miltiorrhiza* and *Panax notoginseng* herbal pair. Modern pharmacological studies have shown that saponins, phenylpropanoids, and terpenoids all possess cardiovascular protective, immunomodulatory, anti-inflammatory, antioxidant, and antitumor activities, consistent with the traditional uses of the *Salvia miltiorrhiza* and *Panax notoginseng* herbal pair for promoting blood circulation, removing blood stasis, clearing the channels, and relieving pain. Therefore, it can be inferred that these components are also the main active ingredients of the *Salvia miltiorrhiza* and *Panax notoginseng* herbal pair; this result also provides some support from a material basis perspective for the synergistic mechanism of the *Salvia miltiorrhiza* and *Panax notoginseng* herbal pair.

[0060] II. Chemical Constituent Analysis of Danshen and Panax notoginseng Pairs Based on Offline 2D-LC-Q-Orbitrap HRMS

[0061] In summary, this example has established a UPLC-Q-Orbitrap HRMS method for the chemical composition analysis of the *Salvia miltiorrhiza* and *Panax notoginseng* herb pair, and preliminarily identified 119 compounds. However, due to the complex composition of the *Salvia miltiorrhiza* and *Panax notoginseng* herb pair, and the relatively low peak capacity and limited separation ability of conventional one-dimensional liquid chromatography methods, the identification results also show that some compounds were not well separated, which may lead to a lack of comprehensiveness in the study of the chemical composition of the *Salvia miltiorrhiza* and *Panax notoginseng* herb pair. Therefore, based on the identification results of UPLC-Q-Orbitrap HRMS, this section uses an offline 2D-LC-Q-Orbitrap HRMS method to collect information such as retention time and characteristic ion fragments of the chemical components of the *Salvia miltiorrhiza* and *Panax notoginseng* herb pair in different dimensions, and combines existing literature and reference standard information to conduct in-depth and comprehensive characterization of its chemical components.

[0062] 1. Experimental Materials

[0063] The samples used were the same as those in Part 1. In addition, the following experimental instruments were used: a semi-preparative liquid chromatograph (model LC-20A, Shimadzu Instruments (Suzhou) Co., Ltd.), a high-speed Chinese medicine pulverizer (model HS-500A, Xi'an Hardware and Medicine Factory, Yongkang City, Zhejiang Province), a high-speed refrigerated centrifuge (model 5425R, Eppendorf (Shanghai) International Trading Co., Ltd.), a nitrogen generator (model BIO-CAD, Shanghai Xiwei Medical Technology Co., Ltd.), and a dry nitrogen blowing device (model MD 200-2, Shanghai Huxi Industrial Co., Ltd.). The information on the chromatographic columns used is shown in the table below.

[0064] Table 4. Chromatographic Column Information

[0065]

[0066]

[0067] 2. Methods and Results

[0068] 2.1 Basic Principles of Offline Two-Dimensional Liquid Chromatography

[0069] 2D-LC is a high-efficiency separation system based on chromatographic columns with two orthogonal separation modes connected in series. Figure 13Depending on whether the sample directly proceeds to the next dimension after separation in the first dimension, 2D-LC can be divided into online and offline modes. In offline mode, the components separated in the first dimension are first collected, then concentrated and reconstituted, before being injected into the second dimension chromatogram for further separation, which uses a different separation mechanism than the first dimension. Compared to online mode, offline 2D-LC, with its enrichment and concentration of samples, facilitates the detection of trace components. Furthermore, the column type, mobile phase, flow rate, and other parameters for both dimensions can be independently selected and optimized, eliminating compatibility issues associated with online coupling. Therefore, offline 2D-LC can employ different stationary phases with varying separation mechanisms based on different separation objectives, including reversed-phase liquid chromatography (RPLC), normal-phase liquid chromatography (NPLC), hydrophilic interaction chromatography (HILIC), ion-exchange chromatography (IEC), and molecular exclusion chromatography (MEC), significantly improving peak capacity and separation capability, making it particularly suitable for separating complex samples. Reversed-phase / reversed-phase chromatography, normal-phase / reversed-phase chromatography, hydrophilic / reversed-phase chromatography, and molecular exclusion / reversed-phase chromatography are common combinations of offline two-dimensional systems. Among these, HILIC columns are suitable for separating strongly polar compounds, while RPLC columns are suitable for separating non-polar and weakly polar compounds. Therefore, the HILIC×RPLC combination can effectively separate complex compounds with significant differences in chemical properties and is widely used in the characterization and identification of traditional Chinese medicine components.

[0070] 2.2 Construction of Offline Two-Dimensional Liquid Phase System

[0071] 2.2.1 Column selection and preliminary investigation of mobile phase

[0072] Column and mobile phase parameters are core elements affecting the separation capability of a chromatographic system; their selection directly determines the separation effect. In a two-dimensional liquid chromatography system, the sample first passes through... 1 After separation on a D column, it then enters... 2 D was further separated and the compounds were characterized by mass spectrometry. Because 2 The direct coupling of D-chromatography with mass spectrometry is crucial to the quality of mass spectrometry data, therefore it is necessary to first perform D-chromatography. 2Selection of chromatographic columns. Octadecylsilane-bonded silica gel (C18) columns are widely used in the study of *Salvia miltiorrhiza* and *Panax notoginseng* due to their high separation efficiency, stable packing material, and durability. Furthermore, C18 columns have good compatibility with mass spectrometry, providing abundant data, and are often used in offline two-dimensional liquid chromatography-mass spectrometry systems. 2 D-column. Therefore, this experiment selected seven different types of C18 columns as... 2 Column D was screened to examine its separation performance of 19 mixed reference standards of Danshen and Sanqi. Candidate columns included Hypersil C18, ACE C18, C18-PFP, C18-AR, Super C18, C18-Amide, and C18-AQ. For the first-dimensional column, four columns with different selectivity from the C18 column were selected for evaluation: Xbrigde Amide, GOWON Amide, PFPP, and CN-ES. Column specifications are shown in Table 4. The differences in chromatographic separation performance when methanol and acetonitrile were used as the organic phase were also investigated.

[0073] 2.2.1.1 Chromatographic conditions

[0074] Mobile phase: methanol (A)-0.1% formic acid solution (B) or acetonitrile (A)-0.1% formic acid solution (B); gradient elution; column temperature: 25℃; injection volume: 5 μL; detection wavelength: 203 nm and 270 nm. The gradient elution program and flow rate settings vary depending on the properties and specifications of the column packing material. The flow rate for ACE C18, C18-PFP, C18-AR, Super C18, and C18-Amide is 1 mL / min; for Xbrigde Amide and GOWON Amide, it is 0.8 mL / min; for Hypersil C18 and PFPP, it is 0.3 mL / min; and for C18-AQ and CN-ES, it is 0.2 mL / min.

[0075] Xbrigde Amide and GOWON Amide are both amino columns, and their bonded groups are sensitive to moisture. Therefore, the proportion of water in the mobile phase should not be too high. So gradient elution program 1 (Table 5) is used; the other 9 columns are gradient elution program 2 (Table 6).

[0076] Table 5 Gradient elution program 1

[0077]

[0078] Table 6 Gradient elution program 2

[0079]

[0080] 2.2.1.2 Evaluation of different column combinations and mobile phases

[0081] The retention times of 19 reference standards were recorded on the above 11 chromatographic columns and under two different mobile phase systems (methanol-0.1% formic acid water and acetonitrile-0.1% formic acid water). The results are shown in Tables 7 and 8. The retention times in the tables were then calculated using formula (1) to obtain the normalized retention time (t). R,norm(i) ).

[0082]

[0083] Among them, t R(i) Represents the retention time of each compound, t R,max and t R,min These represent the retention times of the compounds with the longest and shortest retention times, respectively. R,norm(i) The calculation results are shown in Tables 9 and 10.

[0084] Table 7. Retention times (minutes) of 19 reference standards in different chromatographic columns (mobile phase: methanol-0.1% formic acid water)

[0085]

[0086]

[0087] Note: / indicates that the component was not clearly detected due to overlap with other peaks.

[0088] Table 8. Retention times (minutes) of 19 reference standards in different chromatographic columns (mobile phase: acetonitrile-0.1% formic acid aqueous solution)

[0089]

[0090] Note: / indicates that the component was not clearly detected due to overlap with other peaks. GOWON Amide could not determine the retention times of 10 reference standards for Danshen due to severe peak overlap.

[0091] Table 9. Normalized retention times of 19 reference standards in different chromatographic columns (mobile phase: methanol-0.1% formic acid water)

[0092]

[0093] Note: / indicates that the component was not clearly detected due to overlap with other peaks.

[0094] Table 10 Normalized retention times of 19 reference standards in different chromatographic columns (mobile phase: acetonitrile-0.1% formic acid water)

[0095]

[0096]

[0097] Note: / indicates that the component was not clearly detected due to overlap with other peaks.

[0098] Nineteen reference standards were used in four samples. 1 t of D chromatographic column R,norm(i) The X-axis is represented by 7 lines. 2 t of D chromatographic column R,norm(i) Plot 2D scatter plots with the Y-axis as the axis, and calculate the linear regression correlation coefficient (R²). 2 The calculation results are shown in Tables 11 and 12. The orthogonality of the combinations was evaluated by comparing the linear regression correlation coefficients of the normalized retention times of the 19 reference standards with different column combinations. 2 The higher the value, the more similar the retention behavior of the two columns; R 2 The smaller the value, the more dispersed the distribution of each reference standard under this column combination, and the better the orthogonality of the system.

[0099] The results in Tables 11 and 12 show that regardless of whether the mobile phase is methanol (A)-0.1% formic acid water (B) or acetonitrile (A)-0.1% formic acid water (B), 1 When the D column is an amino column (Xbrigde Amide or GOWON Amide), R 2 The value is significantly smaller. However, when the organic phase is methanol, 1 The chromatogram of D shows very poor separation. Figures 5A to 5D Therefore, acetonitrile was chosen as the organic phase. When the mobile phase is acetonitrile (B) - 0.1% formic acid water (B), 1 The D column is an Xbrigde Amide column. 2 When column D is a Hypersil C18, R 2 The value is 0.0473, indicating that the system has good orthogonality and good chromatographic separation. Therefore, considering both orthogonality and chromatographic separation performance, acetonitrile (A)-0.1% formic acid water (B) was finally selected as the mobile phase, and Xbrigde Amide as the... 1 D-column, Hypersil C18 as... 2 A 2D-LC system consisting of HILIC and RPLC was constructed using a D-column.

[0100] Table 11 1 D-column and 2 Orthogonality test results of D-column R 2 (Mobile phase: methanol-0.1% formic acid water)

[0101]

[0102] Table 121 D-column and 2 Orthogonality test results of D-column R 2 (Mobile phase: acetonitrile-0.1% formic acid water)

[0103]

[0104] 2.2.2 1 D Chromatographic conditions investigation

[0105] Under section 2.2.1.2 of this part, it has been determined that... 1 The D-column was an Xbrigde Amide (150×4.6mm, 3.5μm) with acetonitrile as the organic phase and formic acid as the aqueous phase. The concentration of the mobile phase additive, flow rate, column temperature and gradient elution program will be investigated and optimized in the future.

[0106] 2.2.2.1 Investigation of the mobile phase

[0107] The effect of adding different concentrations (0.05%, 0.1%, and 0.2%) of formic acid to the aqueous phase on chromatographic separation was investigated. The results are as follows: Figure 6A and Figure 6B The chromatograms showed that when the formic acid concentration was 0.2%, the baseline drift at 203 nm was significant; however, when the formic acid concentrations were 0.05% and 0.1%, the chromatographic separation effect was not significantly different. Therefore, the choice of mobile phase was determined by comparing the total number of peaks and the total peak area at 203 nm and 270 nm (see Table 13). The results showed that when the formic acid concentration was 0.05%, the total number of chromatographic peaks was the highest and the total peak area was the largest, and the baseline at 203 nm was more stable. Therefore, the mobile phase was finally determined to be acetonitrile (A)-0.05% formic acid solution (B).

[0108] Table 13 Total number of peaks and total peak area of ​​chromatograms with different formic acid concentrations

[0109]

[0110] 2.2.2.2 Investigation of column temperature

[0111] Changes in column temperature not only alter the retention times of chromatographic peaks but also affect analytical efficiency. This experiment investigated the effects of column temperatures of 25℃, 30℃, 35℃, and 40℃ on the separation of various chromatographic peaks in the test solution of *Salvia miltiorrhiza* and *Panax notoginseng*. (See attached figures.) Figure 7A and Figure 7B The results showed that the chromatographic peak separation was not significantly different at different column temperatures, with 25℃ showing a slightly better separation effect than other column temperatures. Considering that higher column temperatures might affect the lifespan of the chromatographic column and increase the risk of equipment failure, 25℃ was ultimately selected as the column temperature.

[0112] 2.2.2.3 Examination of Flow Velocity

[0113] Flow rate is one of the factors affecting chromatographic separation. This experiment investigated the separation of samples at three different flow rates: 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min. Figure 8A and Figure 8B The results showed that the chromatographic peak shape was optimal and the separation effect was good when the flow rate was 0.8 mL / min. Therefore, the flow rate was finally determined to be 0.8 mL / min.

[0114] 2.2.2.4 1 Determination of D-chromatographic conditions

[0115] After the above optimization, the final chromatographic conditions were determined as follows: column: Xbrigde Amide (150×4.6mm, 3.5μm); mobile phase: acetonitrile (A)-0.05% formic acid water (B), with gradient elution according to Table 14; detection wavelengths: 203nm and 270nm; column temperature: 25℃; flow rate: 0.8mL / min; injection volume: 10μL.

[0116] Table 14 1 D-gradient elution process

[0117]

[0118] 2.2.3 2 D Chromatographic conditions investigation

[0119] Under section 2.2.1.2 of this part, it has been determined that... 2 The D column is a Hypersil C18 (100×2.1mm, 1.9μm), the same column used in "Part 1". Therefore, the mobile phase, column temperature, flow rate, and injection volume are the same as in "Part 1".

[0120] After further optimization of the elution gradient, it was determined that... 2 Chromatographic conditions D were as follows: column: Hypersil C18 (2.1 mm × 100 mm, 1.9 μm); mobile phase: acetonitrile (A) - 0.1% formic acid water (B); detection wavelengths: 203 nm and 270 nm; gradient elution according to Table 15; column temperature: 30 °C; flow rate: 0.3 mL / min; injection volume: 3 μL.

[0121] Table 15 2 D-gradient elution process

[0122]

[0123] 2.3 Chemical composition analysis of the Danshen-Sanqi herb pair

[0124] 2.3.1 Chromatographic conditions: 1 Chromatographic conditions: Column: Xbrigde Amide (150×4.6mm, 3.5μm); Mobile phase: acetonitrile (A) - 0.05% formic acid water (B); Detection wavelengths: 203nm and 270nm; Gradient elution according to Table 14; Column temperature: 25℃; Flow rate: 0.8mL / min; Injection volume: 10μL.

[0125] 2 Chromatographic conditions: Column: Hypersil C18 (2.1 mm × 100 mm, 1.9 μm); Mobile phase: acetonitrile (A) - 0.1% formic acid water (B); Gradient elution according to Table 15; Column temperature: 30 °C; Flow rate: 0.3 mL / min; Injection volume: 3 μL.

[0126] 2.3.2 Mass spectrometry conditions: Same as the mass spectrometry conditions in "Part 1".

[0127] 2.3.3 Preparation of test solution: 1 Preparation of the test solution for the D-Dan Shen San Qi herb pair: The preparation method is the same as in "Part One".

[0128] 2 Preparation of the fractional test solution for the Danshen-Sanqi herb pair: In this example, the first dimension was separated using hydrophilic interaction liquid chromatography on a Shimadzu LC-20A semi-preparative liquid chromatography system under the following chromatographic conditions: column: Xbrigde Amide (150×4.6mm, 3.5μm); mobile phase: acetonitrile (A) - 0.05% formic acid water (B); detection wavelengths: 203nm and 270nm; gradient elution according to Table 14; column temperature: 25℃; flow rate: 0.8mL / min; injection volume: 10μL. Twenty injections were repeated under the above chromatographic conditions. Fractional collection was performed based on the peak elution. Figure 9 The 20 fractions were divided into 20 fractions: 0–5 min (fraction 1), 5–9 min (fraction 2), 9–13 min (fraction 3), 13–18 min (fraction 4), 18–22 min (fraction 5), 22–26 min (fraction 6), 26–31 min (fraction 7), 31–35 min (fraction 8), 35–40 min (fraction 9), and 40–45 min (fraction 10). The 20 fractions were mixed sequentially, dried under nitrogen at 60°C, and then reconstituted with 500 μL of 70% ethanol. After centrifugation at 14500 rpm for 10 min, the supernatant was collected as the final fraction. 2 D. Sample to be tested.

[0129] 2.3.4 Preparation of mixed reference solution: Same as in "Part 1".

[0130] 2.3.5 Sample determination: Take... 1 D. The test solution of the Danshen-Sanqi herb pair was prepared according to...1 Analyze under D chromatographic conditions, collect and process the fraction according to the method described in section 2.3.3 of this part, and then proceed as follows: 2 The analysis was performed under D-chromatographic conditions and MS detection was performed.

[0131] 2.3.6 Peak capacity and orthogonality evaluation: 1 Peak capacity of D ( 1 n grd )and 2 Peak capacity of D ( 2 n grd ) can be obtained separately by formula (1), where t g W represents the effective gradient time; b The average peak width of the selected chromatographic peak. The theoretical peak capacity (n) of the constructed offline two-dimensional chromatography system. 2D Then it is 1 D and 2 The product of the D peak capacity is obtained by formula (2). 1 D and 2 The average peak widths of D were 0.46 min and 0.26 min, respectively, and their effective gradient times were 45 min and 35 min, respectively. According to the peak capacity calculation formulas (2) and (3), the peak capacity of the two-dimensional system was calculated to be as high as 13169. This result shows that compared with the traditional one-dimensional liquid chromatography, the peak capacity of the offline 2D-LC system is significantly improved, which is beneficial for the separation and characterization of more chemical components.

[0132] n grd ≈t g / W b (2); n 2D = 1 n grd × 2 n grd (3);

[0133] The orthogonality (O) of the constructed two-dimensional liquid chromatography system was evaluated using the star-striped equation method proposed by Camenzuli. To obtain stable and reliable orthogonality evaluation results, 19 reference standards were selected as evaluation indicators. The retention times of the 19 reference standards in both dimensions were recorded, and their t values ​​were calculated according to formula (1). R,norm(i) The m / z and two dimensions of the control are t. R,norm(i) See Table 16. Use four intersecting lines (Z). － Z + The space is separated by Z1 and Z2, S Z－ S Z+ S Z1 S Z2The distributions in four different directions are represented, and the diffusion of the 19 components around these four lines is determined by taking the standard deviation of these distances as shown in formulas (4) to (7) (where σ is the standard deviation). The calculated values ​​are input into formulas (8) to (11) to obtain four Z parameters, and the orthogonality (A0) value is calculated according to formula (12). The final calculation result shows that the orthogonality is 59% ( Figure 10 and Figure 11 The result indicates that the system has good orthogonality.

[0134]

[0135] Table 16 Information on the 19 control standards used for orthogonality evaluation

[0136]

[0137]

[0138] 2.3.7 Results and Analysis

[0139] This section presents the first offline 2D-LC-Q-Orbitrap HRMS technology for the chemical composition analysis of a Salvia miltiorrhiza and Panax notoginseng sample solution extracted by ultrasonication with a 70% ethanol solution. All 10 fractions showed good response in both positive and negative ion detection modes. The total ion chromatograms of the 10 fractions in positive ion mode are shown below. Figures 12A to 12J Based on the obtained information such as compound retention time, precise relative molecular mass, and secondary fragment ions, and referring to the identification results in "Part I," combined with comparisons with reference standards and literature, 139 compounds were successfully identified, including 59 saponins, 25 terpenoids, 25 phenylpropanoids, 7 flavonoids, and 23 other compounds. The results are shown in Table 17. Among them, 19 compounds were definitively identified by comparing their retention times and mass spectrometry data with reference standards. Compared with the identification results of UPLC-Q-Orbitrap HRMS, 25 new components were identified, mainly highly polar saponins and organic acids, which showed weak retention on the C18 column. This result indicates to some extent that HILIC and RPLC can enhance the analytical capabilities for complex samples through complementary separation mechanisms.

[0140] Table 17. Identification results of chemical components of the *Salvia miltiorrhiza* and *Panax notoginseng* herb pairs based on offline 2D-LC-Q-Orbitrap HRMS.

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147] Note: a Confirmation by comparison with a reference standard; b Newly identified components compared to "Chapter Two": PPD: protopanaxadiol type; PPT: protopanaxadiol type; OA: oleanolic acid type; OT: octyl esteron type; Glc: glucosyl; Xyl: xylose type; Rha: rhamnose type; Mal: ​​malonyl substituent; Ace: acetyl group.

Claims

1. A method for the analysis of chemical components of *Salvia miltiorrhiza* and *Panax notoginseng* based on offline two-dimensional liquid chromatography coupled with mass spectrometry, characterized in that... Includes the following steps: Step (1), Preparation of mixed reference solution: Using methanol as solvent, prepare a mixed reference solution from the reference standard; Step (2), Preparation of one-dimensional chromatographic test solution: The one-dimensional chromatographic test solution was prepared by ultrasonic extraction of the Danshen and Sanqi herb pairs; Step (3), Preparation of two-dimensional chromatographic test solution: Hydrophilic interaction liquid chromatography is used as the first dimension of chromatography. The one-dimensional chromatographic test solution is prepared and separated in a semi-preparative liquid chromatography system. The fractions are collected in segments according to the peaks. Each fraction is dried by nitrogen gas and then redissolved in an ethanol aqueous solution. Finally, the redissolved liquid is centrifuged and the supernatant is taken to obtain the two-dimensional chromatographic test solution. Step (4): The mixed reference solution and the two-dimensional chromatographic test solution obtained in step (3) are separated by reversed-phase chromatography as the second dimension on an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry system and detected by mass spectrometry. The detection results are compared and analyzed to achieve qualitative analysis of chemical components in the Danshen-Sanqi drug pair.

2. The method for chemical component analysis of *Salvia miltiorrhiza* and *Panax notoginseng* based on offline two-dimensional liquid chromatography coupled with mass spectrometry according to claim 1, characterized in that, In step (1), the reference standards include quercetin, notoginsenoside R1, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Rg1, 20(S)-ginsenoside Rg3, ginsenoside Re, salvianolic acid B, salvianolic acid C, dihydrotanshinone I, salvianolic acid A, tanshinone IIA, tanshinone, tanshinone I, cryptotanshinone, sodium tanshinone, and rosmarinic acid.

3. The method for chemical component analysis of *Salvia miltiorrhiza* and *Panax notoginseng* based on offline two-dimensional liquid chromatography coupled with mass spectrometry according to claim 2, characterized in that... In step (1), the mixed reference solution contains quercetin at a concentration of 70-80 μg / mL, notoginsenoside R1 at a concentration of 180-200 μg / mL, ginsenoside Rb1 at a concentration of 100-110 μg / mL, ginsenoside Rb2 at a concentration of 90-100 μg / mL, ginsenoside Rc at a concentration of 130-140 μg / mL, ginsenoside Rd at a concentration of 160-170 μg / mL, ginsenoside Rg1 at a concentration of 90-100 μg / mL, 20(S)-ginsenoside Rg3 at a concentration of 90-100 μg / mL, and ginsenoside Re at a concentration of 80 μg / mL. The concentrations of the following ingredients are as follows: -90 μg / mL, 135-145 μg / mL for salvianolic acid B, 215-225 μg / mL for salvianolic acid C, 65-75 μg / mL for dihydrotanshinone I, 80-90 μg / mL for salvianolic acid A, 60-70 μg / mL for tanshinone IIA, 70-80 μg / mL for tanshinone neoketone, 70-80 μg / mL for tanshinone I, 170-180 μg / mL for cryptotanshinone, 180-190 μg / mL for sodium tanshinone, and 90-100 μg / mL for rosmarinic acid.

4. The method for chemical component analysis of *Salvia miltiorrhiza* and *Panax notoginseng* based on offline two-dimensional liquid chromatography coupled with mass spectrometry according to claim 1, characterized in that, In step (2), the solvent used for ultrasonic extraction is a methanol-water solution or an ethanol-water solution, with a methanol volume fraction of 50-80% and an ethanol volume fraction of 50-80% in the methanol-water solution; during ultrasonic extraction, the ultrasonic power is 100-200W, the ultrasonic frequency is 30-50kHz, and the ultrasonic treatment time is 20-40min; the mass ratio of Panax notoginseng powder to Panax notoginseng powder in the Panax notoginseng-Salvia miltiorrhiza herb pair is 1:(0.5-2); the mass-volume ratio of the Panax notoginseng-Salvia miltiorrhiza herb pair to the solvent is 0.03-0.05g / mL; both Panax notoginseng powder and Panax notoginseng powder pass through a 65-mesh sieve.

5. The method for chemical component analysis of *Salvia miltiorrhiza* and *Panax notoginseng* based on offline two-dimensional liquid chromatography coupled with mass spectrometry according to claim 4, characterized in that... In step (2), the solvent used for ultrasonic extraction is an aqueous ethanol solution with a volume fraction of 70%; the ultrasonic power is 150W, the ultrasonic frequency is 40kHz, and the ultrasonic treatment time is 30min; the mass ratio of Panax notoginseng powder to Panax notoginseng powder in the Panax notoginseng-Panax notoginseng herb pair is 1:1; the mass-volume ratio of the Panax notoginseng-Panax notoginseng herb pair to the solvent is 0.04g / mL.

6. The method for chemical component analysis of *Salvia miltiorrhiza* and *Panax notoginseng* based on offline two-dimensional liquid chromatography coupled with mass spectrometry according to claim 1, characterized in that, In step (3), the semi-preparative liquid chromatography system used in the first dimension chromatography is model LC-20A; in step (4), in the ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry system, the high-resolution mass spectrometer is model Oribitrap 120 and the ultra-high performance liquid chromatograph is model Vanquish core.

7. The method for chemical component analysis of *Salvia miltiorrhiza* and *Panax notoginseng* based on offline two-dimensional liquid chromatography coupled with mass spectrometry according to claim 6, characterized in that... In step (3), the chromatographic conditions for the first dimension chromatography are as follows: the hydrophilic interaction liquid chromatography column is an Xbrigde Amide column with a size of 150×4.6mm and a packing particle size of 3.5μm; the mobile phase A is acetonitrile, and the mobile phase B is 0.05% formic acid water, i.e., 0.5mL formic acid is diluted to 1L with water; the detection wavelengths are 203nm and 270nm; the column temperature is 25℃; the flow rate is 0.8mL / min; and the injection volume is 10μL. The elution program for the first-dimensional chromatography is as follows: 0-10 min, mobile phase A volume fraction 95%; 10-15 min, mobile phase A volume fraction 95%-90%; 15-20 min, mobile phase A volume fraction 90%-80%; 20-30 min, mobile phase A volume fraction 80%-75%; 30-40 min, mobile phase A volume fraction 75%-70%; 40-45 min, mobile phase A volume fraction 70%-65%.

8. The method for chemical component analysis of *Salvia miltiorrhiza* and *Panax notoginseng* based on offline two-dimensional liquid chromatography coupled with mass spectrometry according to claim 7, characterized in that... In step (3), the fractions collected in segments are: 0-5min as fraction 1, 5-9min as fraction 2, 9-13min as fraction 3, 13-18min as fraction 4, 18-22min as fraction 5, 22-26min as fraction 6, 26-31min as fraction 7, 31-35min as fraction 8, 35-40min as fraction 9, and 40-45min as fraction 10. The injection is repeated 15-25 times. The fractions collected in segments from each injection are mixed according to the fraction number and then dried with nitrogen and reconstituted.

9. The method for chemical component analysis of *Salvia miltiorrhiza* and *Panax notoginseng* based on offline two-dimensional liquid chromatography coupled with mass spectrometry according to claim 8, characterized in that, In step (3), when each fraction is dried with nitrogen, the temperature of the drying nitrogen is 50-70℃; the volume fraction of ethanol in the ethanol aqueous solution used for redissolution is 60-80%; the centrifugation rate is 14000-15000 rpm and the centrifugation time is 10-15 min.

10. The method for chemical component analysis of *Salvia miltiorrhiza* and *Panax notoginseng* based on offline two-dimensional liquid chromatography coupled with mass spectrometry according to any one of claims 1-9, characterized in that, In step (4), the chromatographic conditions for the second dimension chromatography are as follows: the reversed-phase column is a Hypersil C18 column with a specification of 100×2.1mm and a packing particle size of 1.9μm; mobile phase A is acetonitrile, and mobile phase B is 0.1% formic acid water, i.e., 1.0mL formic acid is diluted to 1L with water; the detection wavelengths are 203nm and 270nm; the column temperature is 30℃; and the flow rate is 0.3mL / min. Injection volume: 3 μL; The elution program for the second-dimensional chromatography is as follows: 0-3 min, mobile phase A volume fraction 8%; 3-5 min, mobile phase A volume fraction 8%-20%; 5-15 min, mobile phase A volume fraction 20%-25%; 15-20 min, mobile phase A volume fraction 25%-45%; 20-25 min, mobile phase A volume fraction 45%-65%; 25-30 min, mobile phase A volume fraction 65%-90%; 30-35 min, mobile phase A volume fraction 90%-95%.

Images