Multi-component characterization method of Jichuan decoction and application of multi-component characterization method

Abstract

The invention relates to the technical field of Chinese patent medicine analysis, in particular to a multi-component characterization method of Jichuan decoction and application of the multi-component characterization method. According to the multi-component characterization method provided by the invention, an off-line two-dimensional liquid chromatography tandem quadrupole-electrostatic field orbitrap high-resolution mass spectrometry technology is adopted, and efficient and accurate analysis of various chemical components of Jichuan decoction is realized through specific chromatographic conditions and mass spectrometry conditions. By means of the method, more than two hundred compounds can be identified from the Jichuan decoction, the compounds contain flavonoids, terpenoids, phenylethanoid glycosides, organic acids, coumarins, phenylpropanoids, iridoids, phthalides, steroids and other components, and reference can be provided for research of pharmacodynamic substances of the Jichuan decoction.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine analysis, and in particular to a multi-component characterization method of Jichuanjian and application thereof. BACKGROUND

[0002] Jichuanjian has the effects of warming kidney and nourishing essence, moistening intestines and relieving constipation, and is commonly used for treating constipation due to kidney deficiency. According to Jing Yue Quan Shu, Jichuanjian is composed of Angelica sinensis (three to five coins), Radix Hedysari (two to three coins), Alisma orientale (one coin and a half), Cnidium officinale (five to seven parts or one coin), and Fructus Aurantii (one coin). Since Jichuanjian is made of multiple traditional Chinese medicines, each of which contains dozens or even hundreds of chemical components, new components are generated during the processing, and the properties of these chemical components are significantly different. Therefore, the complexity of components makes it difficult to determine the material basis, and also increases the difficulty of quality control and mechanism research. In the analysis of complex samples, chemical analysis methods may be interfered by other coexisting substances, resulting in poor selectivity, and the interfering substances may mask or confuse the signal of the substance to be tested, making accurate analysis difficult. Moreover, for complex samples, chemical analysis detection usually takes a long time and requires more steps and time to complete the analysis process. Therefore, in order to comprehensively study the chemical components of Jichuanjian, it is necessary to establish a more comprehensive multi-component characterization method. SUMMARY

[0003] In view of the above technical problems, the present application provides a multi-component characterization method of Jichuanjian and application thereof. The multi-component characterization method can efficiently and accurately identify more than 200 compounds in Jichuanjian, covering flavonoids, terpenes, phenylethanoid glycosides, organic acids, coumarins, phenylpropanoids, iridoid glycosides, phthalides, and steroids, thereby providing a reference for the research of the effective substances of Jichuanjian.

[0004] To achieve the above-mentioned application purposes, the embodiments of the present application adopt the following technical solutions: The first aspect of the present application provides a multi-component characterization method of Jichuanjian, which uses offline two-dimensional liquid chromatography combined with high-resolution mass spectrometry to detect the chemical components in Jichuanjian. The chromatographic conditions of the first dimension chromatography in the offline two-dimensional liquid chromatography are as follows: Chromatographic column: Waters XAmide chromatographic column with a size of 4.6x150 mm and a particle size of 5 μm; Mobile phase A is water, and mobile phase B is acetonitrile. The linear gradient elution program is as follows:

[0005] Flow rate: 1.0 mL / min; column temperature: 25-40℃; The eluents collected at 0-3.5 min, 3.5-5 min, 5-7 min, 7-9.25 min, 9.25-10.25 min, 10.25-12 min, 12-17 min, 17-21 min, 21-25 min, respectively, were dried, and 50% methanol was added to the dried fractions, respectively, and then centrifuged. v / v The supernatant was used for the second dimension chromatography analysis after methanol dissolution and centrifugation. The chromatographic conditions of the second dimension chromatography in the offline two-dimensional liquid chromatography are as follows: The chromatographic column is a BEH C18 chromatographic column with a size of 2.1*100 mm, 1.7 μm, or a HSST3 chromatographic column with a size of 2.1*100 mm, 1.8 μm, or a CSH C18 chromatographic column with a size of 2.1*100 mm, 1.7 μm. The mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is acetonitrile. v / v The mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is acetonitrile. The gradient elution program is as follows:

[0006] The flow rate is 0.3 mL / min, and the column temperature is 25-40℃.

[0007] In the method, the sample to be tested is first separated by the first dimension chromatography system, and the collected components are then successively injected into the second dimension chromatography system connected with the mass spectrometer for further separation and analysis. By using the above-mentioned mobile phase composition, chromatographic column type and gradient elution program, the resolution and peak capacity can be significantly improved. The second dimension of the offline two-dimensional system is connected with the high-resolution mass spectrometer, the fragment information of the compound can be obtained through the multi-stage mass spectrometry (MS / MS), the structure inference and molecular formula confirmation of the unknown components can be realized, and thus the qualitative analysis with high sensitivity can be realized.

[0008] Preferably, the chromatographic column in the chromatographic conditions of the second dimension chromatography is a BEH C18 chromatographic column with a size of 2.1*100 mm, 1.7 μm.

[0009] Preferably, the column temperature in the chromatographic conditions of the second dimension chromatography is 35℃.

[0010] Preferably, the high-resolution mass spectrometer is a quadrupole-electrostatic field orbitrap high-resolution mass spectrometer.

[0011] Further preferably, the parameters of the mass spectrometer are as follows: spray voltage, -3.0 kV / +3.5 kV; sheath gas flow rate, 35 arb; auxiliary gas flow rate, 10 arb; purge gas flow rate, 0 arb; ion transfer tube temperature is 400℃ and ion source heating temperature is set to 300℃, and Full MS / dd-MS is adopted.2 Scan mode, Full MS scan range m / z was set to 100-1500 with a resolution of 60000; MS 2 Mass spectrometry scan was dynamic mass range with a resolution of 15000; collision-induced dissociation was performed at normalized collision energies of 20 / 40 / 60 V with an isolation window set to 1 m / z Dynamic exclusion time was set to 2 s.

[0012] The second aspect of the present application provides application of the above-mentioned multi-component characterization method in quality research of Jichuan Decoction.

[0013] The third aspect of the present application provides application of the above-mentioned multi-component characterization method in quality control of Jichuan Decoction.

[0014] The present application has the beneficial effect that the present application establishes an Offline 2D-LC / Q-Orbitrap-MS method for characterizing compounds in Jichuan Decoction. The system is configured with HILIC and RPLC chromatographic columns, which have good orthogonality and high theoretical peak capacity. Compared with traditional one-dimensional chromatography, not only the peak capacity is greatly increased, but also trace and trace components are enriched, avoiding being masked by high-abundance compounds. Using the multi-component characterization method provided by the present application, a total of 262 compounds are identified in Jichuan Decoction, including 59 flavonoids, 42 terpenes, 26 phenylethanoid glycosides, 19 organic acids, 18 coumarins, 14 phenylpropanoids, 12 iridoid glycosides, 9 phthalides, 6 steroids, and 57 other types of components. Methodology verification shows that the intra-day and inter-day precision of the system is within 5% RSD, and the repeatability RSD value is less than 10%. This shows that the method constructed by the present application can efficiently and accurately identify the chemical components of the traditional Chinese medicine compound Jichuan Decoction, and can provide a reference for its efficacy material basis, and can be used for quality research or quality control of Jichuan Decoction. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the sample preparation diagram based on the first dimension HILIC in Example 1 of the present application and the total ion current diagram of each fraction of Jichuan Decoction; Figure 2 is the total ion current diagram obtained by different reversed-phase chromatographic columns in negative ion mode and the number of chromatographic peaks detected in negative ion mode in Test Example 1; Figure 3 is the orthogonal evaluation scatter plot of different first dimension chromatographic columns and second dimension BEH C18 chromatographic columns in Test Example 2 of the present application; Figure 4 is the peak shape and separation degree of chromatographic peaks when the first dimension chromatography uses different aqueous phases in Test Example 3 of the present application; Figure 5 Peak shape and separation degree of chromatographic peaks when different column temperatures are used in the first dimension chromatography in Test Example 4 of the present application; Figure 6 Number of ion chromatographic peaks extracted when different water phases are used in the second dimension chromatography separation in Test Example 5 of the present application; Figure 7 Peak shape and separation degree of chromatographic peaks when different column temperatures are used in the second dimension chromatography in Test Example 6 of the present application; Figure 8 Orthogonality result of the off-line two-dimensional liquid chromatography system in Test Example 7 of the present application; Figure 9 Peak area of an index component under different spray voltages in Test Example 8 of the present application; Figure 10 Peak area of an index component under different ion transmission tube temperatures in Test Example 9 of the present application; Figure 11 Peak area of an index component under different ion source heating temperatures in Test Example 10 of the present application; Figure 12 Secondary ion fragments obtained by using different normalized collision energies in the negative ion mode in Q-Orbitrap in Test Example 11 of the present application. DETAILED DESCRIPTION

[0016] In order to make the objectives, technical solutions and advantages of the present application clearer and more comprehensible, the present application will be further described in detail below with specific examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making any creative efforts fall within the protection scope of the present application.

[0017] JiChuanJian is made of six Chinese medicines, and has multiple and complex chemical components and significant differences in properties. It is not easy to comprehensively, accurately and quickly characterize the multiple components by using conventional chemical analysis methods.

[0018] In view of the problem, the embodiment of the present application provides a multi-component characterization method of JiChuanJian. The method detects chemical components in JiChuanJian by using off-line two-dimensional liquid chromatography combined with high-resolution mass spectrometry. Chromatographic conditions of the first dimension chromatography in the off-line two-dimensional liquid chromatography are as follows: A Waters XAmide chromatographic column with a specification of 4.6*150 mm and 5 μm is used as the chromatographic column; Water is used as the mobile phase A, acetonitrile is used as the mobile phase B, and a linear gradient elution program is as follows:

[0019] Flow rate: 1.0 mL / min; column temperature: 25-40℃; The eluents collected at 0-3.5 min, 3.5-5 min, 5-7 min, 7-9.25 min, 9.25-10.25 min, 10.25-12 min, 12-17 min, 17-21 min, 21-25 min were dried respectively after being combined, and then 50% v / v Dissolved in methanol, and the supernatant was used for second-dimensional chromatographic analysis after centrifugation; The chromatographic conditions of the second-dimensional chromatography in the off-line two-dimensional liquid chromatography are as follows: Chromatographic column: BEH C18 chromatographic column with a size of 2.1x100 mm, 1.7 μm; or HSS T3 chromatographic column with a size of 2.1x100 mm, 1.8 μm; or CSH C18 chromatographic column with a size of 2.1x100 mm, 1.7 μm; Mobile phase A: 0.1% v / v Mobile phase B: acetonitrile; The gradient elution program is as follows:

[0020] Flow rate: 0.3 mL / min; column temperature: 25-40℃.

[0021] The application also provides an application of the above multi-component characterization method in quality research or quality control of Jichuan decoction.

[0022] The technical solutions of the application are further described below through specific examples.

[0023] The instruments used in the following examples are as follows: Thermo Ultimate 3000 double ternary ultra-high performance liquid chromatography system, Vanquish ultra-high performance liquid chromatography system, Orbitrap Exploris 240 high-resolution mass spectrometer, all purchased from Thermo Fisher Corporation, USA. Chromatographic columns HSS T3, CSH C18, BEH C18, BEH Shield RP18, Xbridge Amide are all purchased from Waters; Chromatographic columns StarCore Amide, XAmide are all purchased from Acchrom; The reagents and drugs used in the following examples are as follows: 53 reference substances (neochlorogenic acid, ferulic acid, fumaric acid, succinic acid, β-ecdysterone, hesperidin, verbascoside, trilobatin, geniposidic acid, angelica lactone A, ligusticum lactone, ginsenoside Ro, thalictrin, nobiletin, neohesperidin, naringin, apigenin, diosmin, 23-acetyl-zeoridin B, 23-acetyl-zeoridin C, guanosine, uridine, uracil, adenine, mannitol, betaine, glucose, sucrose, trehalose, tyrosine, histidine, arginine, isoleucine, phenylalanine, proline, tryptophan, valine, azelaic acid, caffeic acid, malic acid, citric acid, vanillin, isoferulic acid, chlorogenic acid, gallic acid, ursolic acid, protocatechuic acid, scopolin, salidroside, hesperidin, oleanolic acid, 7-hydroxycoumarin, ostruthin) were purchased from Chengdu Mansite Biotechnology Co., Ltd. HPLC grade acetonitrile, methanol (Fisher, Fair lawn, NJ, USA), formic acid (ACS, Wilmington, DE, USA), acetic acid (Sigma-Aldrich, St. Louis, MO, USA), and water (Vita Water Food & Beverage Co., Ltd., Guangzhou, China); Angelica sinensis (Oliv.) Diels, Radix Angelicae Sinensis, Rhizoma Alismatis, Rhizoma Alismatis, Rhizoma Coptidis, Radix Coptidis, Fructus Aurantii, Fructus Aurantii, and Cultivated Varieties, Radix et Rhizoma Cnidii, Radix et Rhizoma Cnidii, were purchased from Hebei Chun Kai Pharmaceutical Co., Ltd. and were dried roots of the plant Angelica sinensis (Oliv.) Diels, dried roots of the plant Alisma orientalis (Sam.) Juzep, dried tubers of the plant Alisma orientalis (Sam.) Juzep, dried rhizomes of the plant Coptis deltoidea C.Y. Wu et How, dried immature fruits of the plant Citrus aurantium L. and Cultivated Varieties, and dried succulent stems with scales of the plant Cistanche tubulosa Y. C. Ma. Angelica sinensis Diels Achyranthes bidentata Alisma plantago-aquatica Cimicifuga foetida Citrus aurantium L. Cistanche deserticola

[0024] Example 1 This example provides a method for multi-component characterization of Jichuanjian.

[0025] 1. Sample preparation 1.1 Off-line two-dimensional liquid chromatography sample preparation ​​​​​​​Jichuan Decoction 30 (Angelica 447.6 g, Cyathula 223.8 g, Cistanche 279.9 g, Alisma 168 g, Cimicifuga 78.3 g, Citrus aurantium 111.9 g) was soaked in a 20 L round-bottom flask for half an hour, then extracted twice by condensing and refluxing water at a solid-liquid ratio of 1:20. Timing started from the beginning of liquid micro-boiling, and each extraction lasted for 2 h. After combining the two extraction solutions, they were concentrated under reduced pressure. A portion of the concentrated solution was freeze-dried into powder to obtain the water extract of Jichuan Decoction. Each single herb was weighed at 20 g, soaked in a 1 L round-bottom flask for half an hour, then extracted twice by condensing and refluxing water at a solid-liquid ratio of 1:20. Timing started from the beginning of liquid micro-boiling, and each extraction lasted for 2 h. After combining the two extraction solutions, they were concentrated under reduced pressure. A portion of the concentrated solution was freeze-dried into powder to obtain the water extract of each single herb.

[0026] A small amount of the water extract of Jichuan Decoction was precisely weighed and placed in a 1.5 mL centrifuge tube. It was configured into a 50 mg / mL stock solution with 50% methanol. After vortex mixing, it was ultrasonically extracted for 30 min. It was centrifuged at 14000 rpm (4°C) for 10 min, and 5 µL of the supernatant was injected into the first-dimension Ultimate 3000 dual-triple ultra-high performance liquid chromatography system for analysis (50 mg / mL). The sample prepared by segmenting from the first-dimension liquid chromatography (1D-LC), the water extract of Jichuan Decoction (configured into a 10 mg / mL solution with 50% methanol, centrifuged at 14000 rpm, 4°C for 10 min, and 5 µL of the supernatant was taken), and the single herb water extract treated in the same way (10 mg / mL) were injected into the Vanquish ultra-high performance liquid chromatography system coupled with Orbitrap Exploris 240 high-resolution mass spectrometry for analysis.

[0027] 1.2 Preparation of marker solution An appropriate amount of 53 kinds of reference substances was precisely weighed and dissolved in pure methanol (apigenin was dissolved in 50% methanol) to prepare a 1.0 mg / mL stock solution. Each reference substance stock solution was diluted with methanol (apigenin was diluted with 50% methanol) to prepare a mixed reference solution with a mass concentration of about 1 µg / mL for each component.

[0028] 2. Instrument parameters 2.1 Conditions of offline two-dimensional liquid chromatography system 1 D separation was performed on a Thermo Ultimate 3000 dual-triple ultra-high performance liquid chromatography system. Column: XAmide (4.6×150 mm, 5 µm); mobile phase: water (A), acetonitrile (B); column temperature: 35°C; flow rate: 1.0 mL / min; injection volume: 5 µL; elution gradient as shown in Table 1.

[0029] Table 1 Elution gradient for first-dimension HILIC separation

[0030] 2 D Separation was performed on a Vanquish ultra-high performance liquid system. Column: BEH C18 (2.1 x 100 mm, 1.7 μm); mobile phase: 0.1% formic acid in water (A), acetonitrile (B); column temperature: 35 °C; flow rate: 0.3 mL / min; injection volume: 2 μL; elution gradient as shown in Table 2.

[0031] Table 2 Elution gradient for the second dimension RPLC separation

[0032] 2.2 Mass spectrometry conditions Mass spectrometry analysis was performed on an Orbitrap Exploris 240 hybrid Q-Orbitrap mass spectrometer equipped with a heated electrospray ion source (HESI). The parameters were set as follows: spray voltage -3.0 kV / +3.5 kV; sheath gas (N2) flow rate, 35 arb; auxiliary gas (N2) flow rate, 10 arb; sweep gas (N2) flow rate, 0 arb; ion transfer tube temperature was 400 °C and ion source heating temperature was set to 300 °C. Full MS / dd-MS 2 (Top5) scan mode was used in this experiment. The Full MS scan range m / z was 100-1500 with a resolution of 60000; MS 2 spectral scan was dynamic mass range with a resolution of 15000. Collision-induced dissociation (HCD) was performed at a normalized collision energy (NCE) of 20 / 40 / 60 V with an isolation window of 1 m / z . Dynamic exclusion time was set to 2 s to exclude repeated fragment information within 2 s to ensure more effective recording of more fragment information. A targeted mass list was also set to target the determination of complex compounds in Jichuan decoction and exclude the interference of irrelevant ions.

[0033] 3. Methodology investigation 3.1 Precision The intra-day precision is that the same sample solution is precisely sucked under the first and second dimensional chromatographic conditions, the sample is injected for 6 times continuously in one day, and the RSD value of the peak area of 5 chromatographic peaks with good separation is calculated. The inter-day precision is that the same sample solution is injected for three days under the first and second dimensional chromatographic conditions, and the RSD value of the peak area of 5 chromatographic peaks of the compounds selected in the first and second dimensions is calculated. The results are shown in Tables 3 and 4. The RSD values of the intra-day and inter-day precisions of the liquid phase system HILIC-UV and two-dimensional liquid chromatography-mass spectrometry RP-MS are all within 5%, which indicates that the offline two-dimensional liquid chromatography system constructed in the application has good precision.

[0034] Table 3 Intra-day precision of offline two-dimensional liquid chromatography system n =6)

[0035] Table 4 Inter-day precision of offline two-dimensional liquid chromatography system n =3)

[0036] 3.2 Reproducibility The prepared Jichuan decoction stock solution is taken, and 6 sample solutions are prepared in parallel. After the one-dimensional chromatographic fractionation, the sample of Fr. 5 is selected, dried by nitrogen, and redissolved with 50% methanol. Then, the sample is determined for 6 times in the second dimension, and the RSD value of the peak area of 5 compounds with good separation is calculated. The RSD result is within 10%, which indicates that the offline two-dimensional liquid chromatography system constructed in the application has good reproducibility.

[0037] Table 5 Reproducibility of offline two-dimensional liquid chromatography system (n=6)

[0038] 4. Multi-component characterization of Jichuan decoction In the first dimension chromatography, all ingredients of Jichuan Decoction were divided into 9 segments, in turn, 0-3.5 min (Fr.1), 3.5-5 min (Fr.2), 5-7 min (Fr.3), 7-9.25 min (Fr.4), 9.25-10.25 min (Fr.5), 10.25-12 min (Fr.6), 12-17 min (Fr.7), 17-21 min (Fr.8), 21-25 min (Fr.9). After 15 preparations, the same segment fraction was combined, concentrated under reduced pressure, dried with nitrogen, 50 μL 50% methanol was used for re-dissolution, vortexed for 2 min, 14000 rpm high speed centrifugation for 10 min, and the supernatant was used for second dimension analysis, and then mass spectrometry analysis. Through data acquisition, the first dimension HILIC sample preparation map and the total ion chromatogram of each fraction of Jichuan Decoction are shown in Figure 1 .

[0039] The collected data were first processed by Compound Discoverer (CD) software. After CD processing, Xcailbur software was used for processing, according to the elemental composition and mass-to-charge ratio provided by CD, the error was controlled within ±5ppm, and Xcailbur software was used for identification. UHPLC-Q-Orbitrap-MS detected 174 compounds, Offline 2D-LC-MS detected 262 compounds. Among them, 53 compounds were compared with standard products (including retention time, mass number of quasi-molecular ion and secondary fragment information), and the rest of the compounds were identified with Metlin, Massbank, HMDB database and related literature (including mass number of quasi-molecular ion and secondary fragment information).

[0040] Example 2 The embodiment provides a multi-component characterization method of Jichuan Decoction.

[0041] 1, Sample preparation: same as example 1.

[0042] 2, Instrument parameters 2.1 Offline two-dimensional liquid chromatography system conditions: basically the same as example 1, the difference is that the second dimension chromatography column is HSST3 (2.1×100 mm, 1.8 μm).

[0043] 2.2 Mass spectrometry conditions: same as example 1.

[0044] Example 3 The embodiment provides a multi-component characterization method of Jichuan Decoction.

[0045] 1, Sample preparation: same as example 1.

[0046] 2, Instrument parameters 2.1 Off-line two-dimensional liquid chromatography system conditions: essentially the same as in Example 1, except that the second dimension chromatographic column was a CSH C18 (2.1 x 100 mm, 1.7 μm).

[0047] 2.2 Mass spectrometry conditions: same as in Example 1.

[0048] Example 4 This example provides a multi-component characterization method for Jichuan Decoction.

[0049] 1. Sample preparation: same as in Example 1.

[0050] 2. Instrument parameters 2.1 Off-line two-dimensional liquid chromatography system conditions: essentially the same as in Example 1, except that the second dimension chromatographic column was a CSH C18 (2.1 x 100 mm, 1.7 μm).

[0051] 2.2 Mass spectrometry conditions: same as in Example 1.

[0052] Example 5 This example provides a multi-component characterization method for Jichuan Decoction.

[0053] 1. Sample preparation: same as in Example 1.

[0054] 2. Instrument parameters 2.1 Off-line two-dimensional liquid chromatography system conditions: essentially the same as in Example 1, except that the second dimension chromatographic column was a CSH C18 (2.1 x 100 mm, 1.7 μm).

[0055] 2.2 Mass spectrometry conditions: same as in Example 1.

[0056] Example 6 This example provides a multi-component characterization method for Jichuan Decoction.

[0057] 1. Sample preparation: same as in Example 1.

[0058] 2. Instrument parameters 2.1 Off-line two-dimensional liquid chromatography system conditions: essentially the same as in Example 1, except that the second dimension chromatographic column was a CSH C18 (2.1 x 100 mm, 1.7 μm).

[0059] 2.2 Mass spectrometry conditions: same as in Example 1.

[0060] Comparative Example 1 This comparative example provides a multi-component characterization method for Jichuan Decoction.

[0061] 1. Sample preparation: same as in Example 1.

[0062] 2. Instrument parameters 2.1 Off-line two-dimensional liquid chromatography system conditions: same as in Example 1, except that the second dimension chromatographic column was a BEH Shield RP18 (2.1 x 100 mm, 1.7 μm).

[0063] 2.2 Mass spectrometry conditions: same as in Example 1.

[0064] Comparative Example 2 This comparative example provides a multi-component characterization method for Jichuan Decoction.

[0065] 1. Sample preparation: same as in Example 1.

[0066] 2. Instrument parameters 2.1 Off-line two-dimensional liquid chromatography system conditions: same as in Example 1, except that the first dimension chromatographic column was a StarCore Amide or Xbridge Amide.

[0067] 2.2 Mass spectrometry conditions: same as in Example 1.

[0068] Comparative Example 3 This comparative example provides a multi-component characterization method for Jichuan Decoction.

[0069] 1. Sample preparation: same as in Example 1.

[0070] 2. Instrument parameters 2.1 Off-line two-dimensional liquid chromatography system conditions: same as in Example 1, except that the water phase in the first dimension chromatographic conditions was 0.1% formic acid or 0.1% acetic acid.

[0071] 2.2 Mass spectrometry conditions: same as in Example 1.

[0072] Comparative Example 4 This comparative example provides a multi-component characterization method for Jichuan Decoction.

[0073] 1. Sample preparation: same as in Example 1.

[0074] 2. Instrument parameters 2.1 Off-line two-dimensional liquid chromatography system conditions: same as in Example 1, except that the column temperature in the first dimension chromatographic conditions was 30°C or 40°C.

[0075] 2.2 Mass spectrometry conditions: same as in Example 1.

[0076] Comparative Example 5 This comparative example provides a multi-component characterization method for Jichuan Decoction.

[0077] 1. Sample preparation: same as in Example 1.

[0078] 2. Instrument parameters 2.1 Off-line two-dimensional liquid chromatography system conditions: same as in Example 1, except that the aqueous phase in the second dimension chromatography conditions was pure water, 0.1% acetic acid in water, 0.2% formic acid in water.

[0079] 2.2 Mass spectrometry conditions: same as in Example 1.

[0080] Comparative Example 6 This comparative example provides a multi-component characterization method for Jichuan Decoction.

[0081] 1. Sample preparation: same as in Example 1.

[0082] 2. Instrument parameters 2.1 Off-line two-dimensional liquid chromatography system conditions: same as in Example 1, except that the column temperature for the second dimension chromatography was 45°C.

[0083] 2.2 Mass spectrometry conditions: same as in Example 1.

[0084] Comparative Example 7 This comparative example provides a multi-component characterization method for Jichuan Decoction.

[0085] 1. Sample preparation: same as in Example 1.

[0086] 2. Instrument parameters 2.1 Off-line two-dimensional liquid chromatography system conditions: same as in Example 1.

[0087] 2.2 Mass spectrometry conditions: same as in Example 1, except that the spray voltage was 2.5 kV or 3.5 kV.

[0088] Comparative Example 8 This comparative example provides a multi-component characterization method for Jichuan Decoction.

[0089] 1. Sample preparation: same as in Example 1.

[0090] 2. Instrument parameters 2.1 Off-line two-dimensional liquid chromatography system conditions: same as in Example 1.

[0091] 2.2 Mass spectrometry conditions: same as in Example 1, except that the ion transfer tube temperature was 250°C, 300°C, or 350°C.

[0092] Comparative Example 9 This comparative example provides a multi-component characterization method for Jichuan Decoction.

[0093] 1. Sample preparation: same as in Example 1.

[0094] 2. Instrument parameters 2.1 Off-line two-dimensional liquid chromatography system conditions: same as in Example 1.

[0095] 2.2 Mass spectrometry conditions: same as in Example 1, except that the ion source heating temperature was 250 °C, 350 °C, 400 °C or 450 °C.

[0096] Comparative Example 10 This comparative example provides a multi-component characterization method for Jichuan Decoction.

[0097] 1. Sample preparation: same as in Example 1.

[0098] 2. Instrument parameters 2.1 Off-line two-dimensional liquid chromatography system conditions: same as in Example 1.

[0099] 2.2 Mass spectrometry conditions: same as in Example 1, except that the normalized collision energy was 10 / 20 / 30 V, 10 / 20 / 40 V or 10 / 30 / 50 V, respectively.

[0100] Test Example 1 This test example uses UHPLC / Q-Orbitrap-MS to determine the separation degree and peak shape of the main chromatographic peaks in the TIC of the second dimension chromatography in Examples 1-3 and Comparative Example 1, and the number of chromatographic peaks extracted by SEIVE software.

[0101] As shown in Figure 2 , under the same conditions, the BEH C18 chromatographic column has the most extracted number of chromatographic peaks, and exhibits higher separation degree and better peak shape.

[0102] Test Example 2 This test example selects 60 representative components in Jichuan Decoction to evaluate the difference in separation effect of the first and second dimension chromatographic columns in Example 1 and Comparative Example 2, using the relative retention time of the target components on the first and second dimension chromatographic columns as an index.

[0103] First, orthogonal analysis is performed, and then a scatter plot is made (as shown in Figure 3 ), and the correlation coefficient is used to evaluate the orthogonality. The larger the correlation coefficient, the closer the retention times of the first and second dimensions, and the smaller the separation difference; on the contrary, the smaller the correlation coefficient, the greater the difference in retention time between the first and second dimensions, and the better the orthogonality, which can achieve the purpose of two-dimensional separation. The scatter plot analysis results show that the linear regression correlation coefficient of the 60 index components on the XAmide and BEH C18 chromatographic columns is R 2 = 0.2200, indicating that the difference in relative retention time between the two chromatographic columns is large, and the orthogonality is good.

[0104] Test Example 3 The test example uses Thermo Ultimate 3000 liquid phase system to perform first-dimensional chromatographic analysis, and compares the peak shape and separation degree of the chromatographic peaks when different water phases are used in the first-dimensional chromatography in Example 1 and Comparative Example 3.

[0105] As shown in Figure 4 When the water phase is a pure water phase, the peak shape and separation degree of the chromatographic peaks are the best.

[0106] Test Example 4 The test example uses Thermo Ultimate 3000 liquid phase system to perform first-dimensional chromatographic analysis, and compares the peak shape and separation degree of the chromatographic peaks when different column temperatures are used in the first-dimensional chromatography in Example 1 and Comparative Example 4.

[0107] As shown in Figure 5 When the column temperature is 35℃, the peak type is the most and the separation degree is the best.

[0108] Test Example 5 The test example uses Vanquish ultra-high performance liquid phase system to perform second-dimensional chromatographic analysis, and compares the peak shape and separation degree of the chromatographic peaks when different water phases are used in the second-dimensional chromatography in Example 1 and Comparative Example 5.

[0109] As shown in Figure 6 Although the number of ion chromatographic peaks extracted by the SEIVE software is the most and the response is the strongest when acetonitrile-pure water is used as the mobile phase, the peak type and separation degree are not good. At the same time, it is found in the identification process that when acetonitrile-0.1% acetic acid water is used as the mobile phase, the error value (ppm) is larger, and the compound is easy to lose. When acetonitrile-0.1% formic acid water is used, the separation degree and peak type are the best.

[0110] Test Example 6 The test example uses Vanquish ultra-high performance liquid phase system to perform second-dimensional chromatographic analysis, and compares the peak shape and separation degree of the chromatographic peaks when different column temperatures are used in the second-dimensional chromatography in Example 1, Example 4, Example 5, Example 6 and Comparative Example 6.

[0111] As shown in Figure 7 When the column temperature is 35℃, the peak type and separation effect of the chromatographic peaks are the best.

[0112] Test Example 7 The test example investigates the orthogonality and peak capacity of the offline two-dimensional liquid chromatography system of Example 1.

[0113] 1. Orthogonality Select 50 representative target compounds, through a series of asterisk equations, system evaluation each component in two-dimensional chromatographic space distribution characteristics. The calculation formula is shown below. Among them, the normalized retention time of the compound in each chromatographic dimension is calculated by equation (1), and the formula is t R,norm（i） is the normalized retention time of the target compound in the corresponding chromatographic dimension, t I is the retention time of the target compound in the corresponding chromatographic dimension, t D is the dead time, t G is the total effective elution time. Based on the normalized retention time, the distribution of the target compound in the two-dimensional space Z - , Z + , Z1, Z2 four cross lines is evaluated by equations (2)-(5), and the S value is obtained accordingly. Substitute S value into equations (6)-(9) to calculate the corresponding Z parameter. Finally, equation (10) is used to calculate the A0 value to evaluate the orthogonality. (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) Results: After calculation, Z - = 0.87, Z + =0.67, Z1= 0.91, Z2= 0.95, further calculation obtained orthogonality result A0=71%. As Figure 8 shown. This shows that compared with the traditional one-dimensional chromatography, the separation selectivity of the offline two-dimensional liquid chromatography system established by the present application is improved, and more chemical components in Jichuan decoction can be effectively separated and identified.

[0114] 2, peak capacity The theoretical peak capacity (n 2D ) of the two-dimensional liquid chromatography system is the product of the peak capacity (n 1 grd ) of the first dimension chromatography and the peak capacity of the second dimension chromatography (n 2 grd). The specific calculation method is as follows: from the first dimension and the second dimension elution time, one representative chromatographic peak is selected from the front, middle and rear sections, the baseline peak width is measured, and the average peak width value of each dimension is calculated accordingly. Then, the effective elution time of the chromatographic dimension is divided by the average peak width, and the theoretical peak capacity of the dimension is obtained.

[0115] (11) (12) Results: The total effective elution time of the first dimension chromatography is 25 min, the average peak width is 0.55 min, and the peak capacity is calculated to be 46. The total effective elution time of the second dimension chromatography is 30 min, the average peak width is 0.17 min, and the peak capacity is calculated to be 180. Therefore, the peak capacity of the off-line 2D-LC system is 8280. Obviously, compared with the traditional one-dimensional chromatography system, the peak capacity of two-dimensional liquid chromatography is increased by about 180 times, which significantly enhances the separation and characterization ability of complex chemical components in Jichuan decoction, and provides good support for the in-depth analysis of its components.

[0116] Test Example 8 In this test example, uridine (All), ferulic acid (quality control indicator of Angelica in Chinese Pharmacopoeia), ginsenoside Ro (Radix Bistortae), verbascoside (Herba Cistanche), 23-acetyl alisol C (Alisma), isoferulic acid (quality control indicator of Cimicifuga in Chinese Pharmacopoeia) and naringin (Fructus Aurantii) were used as indicator components to investigate the peak areas of the indicator components of Example 1 and Comparative Example 7 under different spray voltages.

[0117] The results are shown in Table 8. Figure 9 The peak areas of the seven indicator components mostly reached the maximum at 3.0 kV.

[0118] Test Example 9 In this test example, uridine, ferulic acid, ginsenoside Ro, verbascoside, 23-acetyl alisol C, isoferulic acid and naringin were used as indicator components to investigate the peak areas of the indicator components of Example 1 and Comparative Example 8 under different ion transfer tube temperatures.

[0119] The results are shown in Table 9. Figure 10 As shown in Table 9, at 400℃, the peak areas of all compounds except uridine reached the maximum.

[0120] Test Example 10 In this test example, uridine, ferulic acid, ginsenoside Ro, verbascoside, 23-acetyl alisol C, isoferulic acid and naringin were used as indicator components to investigate the peak areas of the indicator components of Example 1 and Comparative Example 9 under different ion source heating temperatures.

[0121] The results are shown in Table 10. Figure 11As shown, when the ion source heating temperature is 300℃, most of the compounds have the highest peak area at this time.

[0122] Test Example 11 In this test example, uridine, ferulic acid, ginsenoside Ro, verbascoside, isoferulic acid and naringin were used as index components, and the secondary ion fragments of the index components under different normalized collision energies were investigated. The 23-acetyl alisol C in Alisma orientalis was not detected in the negative ion mode, and the compounds in Alisma orientalis were almost detected in the positive ion mode, so the compounds in Alisma orientalis were not selected.

[0123] As shown in the results, the characteristic fragments of various compounds can be generated under different collision energies, and the ion abundance gradually increases with the increase of the collision energy. When the NCE is set to 20 / 40 / 60 V, the ion abundance is optimal. Figure 12

[0124] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A method for multi-component characterization of Jichuan decoction, characterized in that, The chemical components in Jichuan Decoction are detected by offline two-dimensional liquid chromatography combined with high resolution mass spectrometry; the chromatographic conditions of the first dimension chromatography in the offline two-dimensional liquid chromatography are: The chromatographic column is Waters XAmide chromatographic column with a size of 4.6*150 mm and a thickness of 5 μm; The mobile phase A is water, the mobile phase B is acetonitrile, and the linear gradient elution program is: The flow rate is 1.0 mL / min; and the column temperature is 25-40 ℃; Fractions were collected for 0-3.5 min, 3.5-5 min, 5-7 min, 7-9.25 min, 9.25-10.25 min, 10.25-12 min, 12-17 min, 17-21 min, 21-25 min, and the same fraction was combined and dried, respectively, with 50% methanol solution, and the supernatant was used for the second dimensional chromatographic analysis after centrifugation. v / v Methanol solution, after centrifugation, the supernatant was used for the second dimensional chromatographic analysis; The chromatographic conditions of the second dimension chromatography in the offline two-dimensional liquid chromatography are: The chromatographic column is BEH C18 chromatographic column with a size of 2.1*100 mm and a thickness of 1.7 μm, or HSST3 chromatographic column with a size of 2.1*100 mm and a thickness of 1.8 μm, or CSH C18 chromatographic column with a size of 2.1*100 mm and a thickness of 1.7 μm; Mobile phase A is 0.1% v / v Formic acid in water, mobile phase B is acetonitrile; The gradient elution program is: The flow rate is 0.3 mL / min; and the column temperature is 25-40 ℃.

2. The multi-component characterization method of claim 1, wherein, The chromatographic column in the chromatographic conditions of the second dimension chromatography is BEH C18 chromatographic column with a size of 2.1*100 mm and a thickness of 1.7 μm.

3. The multi-component characterization method of claim 1, wherein, The column temperature in the chromatographic conditions of the second dimension chromatography is 35 ℃.

4. The multi-component characterization method of any one of claims 1-3, wherein, The high resolution mass spectrometry is quadrupole-electric field orbitrap high resolution mass spectrometry.

5. The multi-component characterization method of claim 4, wherein, The parameters of the mass spectrometer were: Spray voltage, -3.0 kV / +3.5 kV; Sheath gas flow rate, 35 arb; Auxiliary gas flow rate, 10 arb; Sweep gas flow rate, 0 arb; Ion transfer tube temperature was 400 °C and ion source heating temperature was set to 300 °C, using Full MS / dd-MS 2 scanning mode, Full MS scan range m / z 100-1500, resolution set to 60000; MS 2 mass spectrum scan was dynamic mass range, resolution was 15000; Collision induced dissociation was performed at normalized collision energy of 20 / 40 / 60 V, isolation window was set to 1 m / z; Dynamic exclusion time was set to 2 s.

6. The multi-component characterization method according to any one of claims 1-5 is applied to the quality research of Jichuan Decoction.

7. The multi-component characterization method according to any one of claims 1-5 is applied to the quality control of Jichuan Decoction.

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