Method for detecting chemical components of Xinnaoxin based on UPLC-Q-TOF-MS

Abstract

The invention relates to the technical field of analysis of chemical components of Xinnaoxin, and particularly discloses a method for detecting chemical components of Xinnaoxin based on UPLC-Q-TOF-MS. The method comprises the following steps: preparing a test solution through alcohol solvent extraction; then the test solution is determined by adopting a UPLC-Q-TOF-MS method, various chemical components of Xinnaoxin can be effectively separated out under specific chromatographic conditions, and the detection sensitivity and accuracy can be improved under specific mass spectrum conditions. 92 kinds of compounds in a heart and brain aoxin sample are finally identified, and the active ingredients of different kinds and different sources have a treatment effect on many heart blood diseases in a synergistic or complementary mode. By adopting the UPLC-Q-TOF-MS method, the chemical components of the Xinnaoxin sample can be rapidly and accurately detected, and reference is provided for promoting secondary development of preparations based on Xinnaoxin and deeper research and development of Xinnaoxin medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical component analysis of Xinnaoxin, and in particular to a method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS. Background Art

[0002] Xinnaoxin (Xinnaoxin) nourishes qi and yin, promotes blood circulation, and removes blood stasis. It is primarily used to treat dizziness, headaches, palpitations, shortness of breath, fatigue caused by qi and yin deficiency and blood stasis, as well as polycythemia caused by hypoxia. Xinnaoxin's main ingredients include natural rhodiola rosea, wolfberry, and fresh seabuckthorn syrup. Rhodiola rosea, a traditional Tibetan medicine, is the main ingredient in the compound, known for its benefits of invigorating qi and promoting blood circulation, dredges the meridians, and relieves asthma. Its main chemical components include salidroside, tyrosol, and gallic acid. Lycium barbarum nourishes the liver and kidneys, improves vital essence, and enhances vision. Research indicates that organic acids such as caffeic acid and p-coumaric acid are closely related to these benefits. Seabuckthorn strengthens the spleen and aids digestion, relieves cough and expectoration, and promotes blood circulation and removes blood stasis. Research suggests that gallic acid and quercetin can serve as quality markers for seabuckthorn's efficacy. However, current quality standards and relevant literature only test for salidroside and betaine, making them difficult to use as comprehensive quality assessment criteria.

[0003] As a compound Chinese medicine composed of three Chinese herbs, Xinnaoxin contains complex chemical components. In the prior art, there are literature reports on a method for determining the content of seven ingredients in Xinnaoxin using a one-test, multiple-evaluation method, which has solved the problem of a single index component. However, the number of drug components detected by this method at one time is still small, and it cannot achieve the purpose of quickly and efficiently comprehensively evaluating the quality of Xinnaoxin drugs. In order to improve the quality control level of Xinnaoxin drugs and at the same time improve the comprehensive evaluation of such drugs, it is urgent to develop a method for detecting all chemical components in Xinnaoxin in a simple, fast, accurate and efficient manner. Summary of the Invention

[0004] In response to the above problems, the present invention provides a method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0006] The method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS comprises the following steps:

[0007] (1) Preparation of test solution: Add the Xinnaoxin sample to an alcohol solvent for extraction, filter, and obtain the test solution;

[0008] (2) The test solution is detected by UPLC-Q-TOF-MS method, wherein:

[0009] Ultra-high performance liquid chromatography (UHPLC) conditions include:

[0010] The chromatographic column is a ZORBAX SB-C18 chromatographic column, the mobile phase A is a 0.1 wt% to 0.5 wt% formic acid aqueous solution, and the mobile phase B is methanol, and gradient elution is performed.

[0011] The conditions of quadrupole time-of-flight tandem mass spectrometry include: electrospray ion source, positive and negative ion modes, and information-correlated acquisition detection mode; wherein, the mass-to-charge ratio scanning range is 100-1500 m / z, the ion source temperature is 520-580°C, the cluster fragmentation voltage is 75-85 V, and the collision energy is 33-37 eV.

[0012] Compared with the prior art, the method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS provided by the present invention first prepares a test solution by extraction with an alcohol solvent; then the test solution is measured by the UPLC-Q-TOF-MS method, and the specific ultra-high performance liquid chromatography conditions can effectively separate the chemical components of Xinnaoxin, and the specific quadrupole time-of-flight tandem mass spectrometry conditions can improve the sensitivity and accuracy of detection. The results of the embodiment show that the present invention finally identified 92 compounds in the Xinnaoxin sample, which respectively belong to flavonoids and their glycosides, organic acids, sugars and glycosides, phenols, amino acids, anthraquinones and their glycosides, terpenes, phenylpropanoids and other compounds. The above active ingredients of different types and sources can have therapeutic effects on many cardiovascular diseases in a synergistic or complementary manner.

[0013] The present invention uses the UPLC-Q-TOF-MS method to quickly and accurately detect the chemical components of Xinnaoxin samples, providing a data basis and scientific basis for its pharmacological material basis and quality control research, and providing a reference for promoting the secondary development of preparations based on Xinnaoxin and for more in-depth research and development of Xinnaoxin drugs.

[0014] Preferably, the alcohol solvent is a methanol aqueous solution or anhydrous methanol with a mass concentration of not less than 50%, more preferably a methanol aqueous solution with a mass concentration of 70% to 80%.

[0015] Preferably, the mass volume ratio of the Xinnaoxin sample to the alcohol solvent is 1g: (17-23)mL.

[0016] The preferred alcohol solvent of the present invention has better solubility and extraction ability for the active ingredients in Xinnaoxin, and the preferred dosage ratio can ensure that no changes or losses of Xinnaoxin components will be caused during the extraction process.

[0017] Preferably, the extraction is ultrasonic extraction with a frequency of 38 to 42 kHz, a power of 250 to 320 W, and an extraction time of 25 to 35 min.

[0018] The preferred ultrasonic extraction conditions of the present invention can further improve the extraction effect.

[0019] Preferably, the filtration is performed using a 0.4-0.5 μm microporous filter membrane.

[0020] Preferably, the ZORBAX SB-C18 chromatographic column has a size of 3.0 mm×100 mm and a filler diameter of 1.8 μm.

[0021] Preferably, the elution procedure of the gradient elution is as follows:

[0022] 0-20 min, 5% → 20% mobile phase A, 95% → 80% mobile phase B;

[0023] 20-70 min, 20% → 60% mobile phase A, 80% → 40% mobile phase B;

[0024] 70-75 min, 60% → 75% mobile phase A, 40% → 25% mobile phase B;

[0025] 75-85 min, 75%→90% mobile phase A, 25%→10% mobile phase B.

[0026] Preferably, the flow rate of the gradient elution is 0.8 to 1.2 mL / min.

[0027] Preferably, the column temperature of the chromatographic column is 25-30°C.

[0028] Preferably, the ultra-high performance liquid chromatography (UHPLC) conditions further include: an injection volume of 8 to 12 μL.

[0029] The preferred ultra-high performance liquid chromatography conditions of the present invention can more effectively separate the various chemical components of Xinnaoxin.

[0030] Preferably, the conditions of the quadrupole time-of-flight tandem mass spectrometry also include: spray voltage of +(5300~5700)V, -(4300~4700)V; nebulizing gas pressure of 53~58psi; curtain gas pressure of 33~37psi; auxiliary gas pressure of 53~58psi; and collision energy rolling range of 13~17eV.

[0031] The preferred conditions of the quadrupole time-of-flight tandem mass spectrometry of the present invention can further improve the sensitivity and accuracy of detection.

[0032] The present invention adopts the UPLC-Q-TOF-MS method, and by screening the conditions of liquid chromatography and mass spectrometry, it realizes the rapid detection of multiple chemical components in Xinnaoxin. The operation is simple, fast, accurate and efficient, the sample pretreatment is simple, the experimental steps are simplified, and the cost is reduced. At the same time, it realizes the rapid qualitative or quantitative analysis of multiple components in Xinnaoxin, which is of great significance for improving the quality standards of Xinnaoxin drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the total ion current diagram of the cerebral isotherm sample in Example 1 of the present invention under positive ion mode;

[0034] Figure 2 This is the total ion current diagram of the cerebral isotherm sample in Example 1 of the present invention under negative ion mode;

[0035] Figure 3 This is the MS pattern of compound 65 in Example 1 of the present invention;

[0036] Figure 4 This is the MS pattern of compound 29 in Example 1 of the present invention;

[0037] Figure 5 This is the MS pattern of compound 23 in Example 1 of the present invention;

[0038] Figure 6 This is the MS pattern of compound 26 in Example 1 of the present invention;

[0039] Figure 7 This is the MS pattern of compound 66 in Example 1 of the present invention;

[0040] Figure 8 This is the MS chart of compound 49 in Example 1 of the present invention. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0042] The drugs and reagents used in the examples of the present invention are shown in Table 1.

[0043] Table 1 Drugs and reagents used in the examples of the present invention

[0044]

[0045]

[0046] The instruments used in the embodiments of the present invention are shown in Table 2.

[0047] Table 2 Instruments used in the embodiments of the present invention

[0048] Instrument name Manufacturer 1290 Infinity UPLC instrument Agilent Technologies, Inc. 6545 Quadrupole Tandem Mass Spectrometer Agilent Technologies, Inc. JJ324BC electronic balance Changshu Shuangjie Testing Instrument Factory Milli-Q water purifier Hebei Antu Trading Co., Ltd. KQ-300DE CNC ultrasonic cleaner Kunshan Ultrasonic Instrument Co., Ltd.

[0049] The Xinnaoxin sample used in the present invention can be Xinnaoxin tablets, Xinnaoxin capsules or Xinnaoxin pills, all of which are composed of three Chinese medicinal herbs: Rhodiola rosea, wolfberry fruit and sea buckthorn juice.

[0050] In order to better illustrate the present invention, further examples are given below.

[0051] Example 1

[0052] Example 1 provides a method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS, comprising the following steps:

[0053] (1) Take 1.00 g of Xinnaoxin sample, grind it into powder in a mortar, pass it through a No. 5 sieve (the inner diameter of the sieve hole is 180±7.6 μm), put it into a conical flask, add 20 mL of 75 wt% methanol aqueous solution and perform ultrasonic extraction for 30 min. The ultrasonic frequency is 40 kHz and the power is 300 W. Cool it to room temperature, make up the lost mass with 75 wt% methanol aqueous solution, and filter it with a 0.45 μm microporous filter membrane to obtain the test solution.

[0054] (2) The test solution is detected by UPLC-Q-TOF-MS method, wherein:

[0055] I. The conditions for ultra-high performance liquid chromatography include:

[0056] The chromatographic column was a ZORBAX SB-C18 column, the mobile phase A was a 0.1 wt % formic acid aqueous solution, and the mobile phase B was methanol, and gradient elution was performed.

[0057] The flow rate was 1.0 mL / min;

[0058] The injection volume was 10 μL;

[0059] The column temperature was 30°C;

[0060] The elution procedure is as follows:

[0061] 0-20 min, 5% → 20% mobile phase A, 95% → 80% mobile phase B;

[0062] 20-70 min, 20% → 60% mobile phase A, 80% → 40% mobile phase B;

[0063] 70-75 min, 60% → 75% mobile phase A, 40% → 25% mobile phase B;

[0064] 75-85 min, 75%→90% mobile phase A, 25%→10% mobile phase B.

[0065] II. The conditions for quadrupole time-of-flight tandem mass spectrometry include:

[0066] Electrospray ionization source (ESI), positive ion mode, detection mode is information-dependent acquisition mode (IDA);

[0067] The mass-to-charge ratio scanning range was 100–1500 m / z;

[0068] The spray voltage (IS) was +5500 V, -4500 V;

[0069] Atomizing gas pressure (GS1) was 55 psi;

[0070] Curtain air pressure (CUR) is 35 psi;

[0071] Auxiliary gas pressure (GS2) is 55psi;

[0072] The ion source temperature (TEMP) was 550°C;

[0073] The cluster lysis voltage (DP) was 80 V;

[0074] The collision energy (CE) was 35 V;

[0075] The collision energy rollover interval (CES) is 15V;

[0076] Multiple mass defect (MMDF) and dynamic background subtraction (DBS) are the conditions for triggering the secondary scan. If these conditions are met, the secondary scan will be performed first.

[0077] The total ion current chromatogram of Xinnaoxin sample is shown in Figures 1-2 , Figure 1 This is the total ion current diagram of the Xinnaoxin sample in positive ion mode. Figure 2 This is the total ion chromatogram of the Xinnaoxin sample in negative ion mode. A total of 30 chemical substances were identified in negative ion mode, and 62 chemical substances were identified in positive ion mode.

[0078] Chemical composition analysis

[0079] Preparation of reference solution: Dissolve salidroside reference, quercetin reference, chlorogenic acid reference, chrysophanol reference, catechin reference and betaine reference in methanol, stir evenly, and obtain methanol solutions containing reference substances (salidroside, quercetin, chlorogenic acid, chrysophanol, catechin and betaine) with a concentration of 20 mg / mL; take 100 μL of each of the above methanol solutions containing reference substances and mix evenly; filter through a 0.22 μm microporous filter membrane to obtain the reference solution.

[0080] It should be noted that the test solution and reference solution obtained in the present invention can be refrigerated (4° C.) for later use as needed.

[0081] The above-mentioned reference solution was detected using the chromatographic conditions and mass spectrometry parameters of the UPLC-Q-TOF-MS method in Example 1. The information library of the chemical components of Xinnaoxin was input into the mass spectrometry analysis software Qualitative AnalysisIF1.7.1 and Peakview 2.2. The peak position search of the target compound was realized by combining the HR-MS / MS Spectral Library1.1 database with the reference substance information. The components corresponding to the target compound were screened out with accurate mass spectrometry (Mass error <5ppm), and secondary cleavage fragment analysis was performed to achieve accurate identification of the chemical components.

[0082] Example 1 92 chemical components in Xinnaoxin were isolated and identified using UPLC-Q-TOF-MS, including flavonoids and their glycosides, organic acids, sugars and their glycosides, phenols, amino acids, anthraquinones and their glycosides, terpenes, phenylpropanoids, and other compounds. Specific results are shown in Table 3.

[0083] Table 3 92 chemical components detected in Xinnaoxin based on UPLC-Q-TOF-MS method

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094] In the table, A, B and C in the “Source” column represent Rhodiola rosea, Lycium barbarum and Hippophae rhamnoides, respectively.

[0095] Example 1 A total of 20 flavonoids were identified from Xinnaoxin. Flavonoids are often combined with sugars to form glycosides. Their fragmentation is mainly RDA fragmentation, but they can also be fragmented by losing sugar groups and neutral fragment ions such as CO, H2O, or C2H2. Taking compound 65 as an example, a quasi-molecular ion peak m / z 303.0500 [M+H] was detected. + The predicted molecular formula is C 15 H 10 O7, whose C ring was cleaved by RDA to obtain a fragment ion m / z 179.0318 [M+H-C8H6O3] + and 121.0276[M+H-C8H6O5] + , then 151.0389[M+H-C8H6O3] was obtained by losing CO and H2O fragment ions. + 、107.0482[M+H-C9H7O5] + 、285.0387[M+H-H2O] + 、257.0433[M+H-H2O-CO] + Compared with the reference substance, it is speculated to be quercetin. The fragmentation pathway is shown in formula 1 and the mass spectrometry analysis is shown in Figure 3 .

[0096]

[0097] Example 1 A total of 26 organic acid compounds were identified from Xinnaoxin. Taking compound 29 as an example, a quasi-molecular ion peak m / z 353.0884 [MH] was detected. - The predicted molecular formula is C 16 H 18 O9, fragment ion m / z 191.0582 [MH-C9H6O3] - 、179.0345[MH-C7H 10 O5] - 、173.0476[MH-C9H6O3-H2O] - and 135.0441[MH-C7H 10 O5-CO2] - Compared with the reference substance, it is speculated to be chlorogenic acid, which is a component contained in wolfberry fruit. The cleavage pathway is shown in formula 2, and the mass spectrometry analysis is shown in Figure 4 .

[0098]

[0099] In Example 1, a total of 7 carbohydrate compounds were identified from Xinnaoxin. Carbohydrate compounds have significant pharmacological effects on various systems of the human body, including the cardiovascular system. Taking compound 23 as an example, a quasi-molecular ion peak m / z 299.1149 [MH] was detected at a retention time of 14.8 min. - The predicted molecular formula is C 14 H 20 O7, fragment ions contain m / z 119.0514 [MH-C6H 13 O6] ﹣ 、137.0949[MH-C6H 11 O5] - It is speculated to be salidroside, the main active ingredient of Rhodiola rosea. The cleavage pathway is shown in formula 3, and the mass spectrometry analysis is shown in Figure 5 .

[0100]

[0101] Example 1 Six phenolic compounds were identified from Xinnaoxin. Phenolic compounds are hydroxyl-containing derivatives of aromatic hydrocarbons. Taking compound 26 as an example, a quasi-molecular ion peak m / z 289.0724 [MH]- was detected at a retention time of 15.71 min, and the predicted molecular formula was C 15 H 14 O6, fragment ion m / z 245.0805 [MH-CO2] ﹣ 、227.0723[MH-CO2-H2O] - 、203.0708[MH-CO2-C2H2O] - 、161.0615[MH-CO2-2(C2H2O)] - 、187.0371[MH-CO2-C3H6O] - 、179.0309[MH-C6H6O2] - , 165.0197 [MH-C7H8O2], 137.0246 [MH-C9H8O3] and 125.0252 [MH-C8H8O3]. Compared with the reference substance, it is speculated to be catechin. Its fragmentation pathway is shown in formula 4, and the mass spectrometry analysis is shown in Figure 6 .

[0102]

[0103] Example 1 Eight quinone compounds were isolated from Xinnaoxin, mainly rhamnolide derivatives among hydroxyanthraquinone derivatives. Taking compound 66 as an example, a quasi-molecular ion peak of m / z 255.0651 [M+H] was detected at a retention time of 48.75 min. + The predicted molecular formula is C 15 H10 O4, fragment ion m / z 227.0245 [M+H-CO] + 、184.7321[M+H-CO-CHO2] + and 156.4302 [M+H-2CO-CH3CO] + It is speculated to be chrysophanol. The mass spectrometry fragmentation pathway is shown in Equation 5. The mass spectrometry analysis diagram is shown in Figure 7 .

[0104]

[0105] Example 1: 8 other compounds were isolated from Xinnaoxin. Taking compound 49 as an example, a quasi-molecular ion peak of m / z 118.0862 [M+H] was detected at a retention time of 36.26 min. + The predicted molecular formula is C5H 11 NO2, by losing carboxymethyl and trimethylamino groups, yields fragment ions m / z 58.0652 [M+H-CH3COOH] + and 59.0730[M+HN(CH3)3] + After comparison with the reference substance, it was speculated to be betaine, the main active ingredient of wolfberry fruit. The cleavage pathway is shown in formula 6, and the mass spectrometry analysis is shown in Figure 8 .

[0106]

[0107] This example uses UPLC-Q-TOF-MS technology, with Xinnaoxin as the research object, and through combined analysis of information such as fragment ions, retention time and relevant literature, a total of 92 compounds were identified, including 20 flavonoids, 26 organic acids, 7 sugars, 6 phenols, 6 amino acids, 8 quinones, 6 terpenes, 5 phenylpropanoids, and 8 other types. It was identified that flavonoids and quinone compounds account for the majority of the components of Rhodiola rosea, and are also the traditional Chinese medicine with the most types of components in Xinnaoxin; both wolfberry and sea buckthorn have the most organic acid compounds. The identified chemical components contain multiple components with significant pharmacological activity, mainly affecting the cardiovascular and cerebrovascular systems. For example, flavonoids are the main active ingredients of the traditional Chinese medicine sea buckthorn, among which kaempferol has a protective effect on the heart and also has antioxidant activity; isorhamnetin, quercetin and myricetin can respectively lower uric acid, relieve liver damage and reduce antibiotic resistance. Salidroside in the carbohydrate compound is the main active ingredient of the traditional Chinese medicine Rhodiola rosea, which can excite myocardial cells and relieve damage caused by myocardial hypoxia. The alkaloid compound betaine is the main active ingredient of the traditional Chinese medicine Lycium barbarum, which has the effect of increasing the amount of protein in the body. In summary, the establishment of UPLC-Q-TOF-MS technology comprehensively characterizes the chemical composition of Xinnaoxin and preliminarily clarifies the material basis of Xinnaoxin, which is of great significance for the subsequent pharmacological research of Xinnaoxin. According to the components of Xinnaoxin, the compounds detected in this example include 34 from Rhodiola rosea, 12 from Lycium barbarum, 15 from Hippophae rhamnoides, and contain 31 partial common compounds.

[0108] It should be noted that as long as the conditions of ultra-high performance liquid chromatography and quadrupole time-of-flight tandem mass spectrometry are within the protection scope defined by the present invention, 92 chemical components (Table 3) in the Xinnaoxin sample can be identified.

[0109] Comparative Example 1

[0110] This comparative example provides a method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS, which is similar to Example 1, except that the extraction solvent in step (1) is acetonitrile, and the other conditions are the same as Example 1. The specific steps are as follows:

[0111] (1) Take 1.00 g of Xinnaoxin sample, grind it into powder in a mortar, pass it through a No. 5 sieve (the inner diameter of the sieve hole is 180±7.6 μm), put it into a conical flask, add 20 mL of acetonitrile and perform ultrasonic extraction for 30 min. The ultrasonic frequency is 40 kHz and the power is 300 W. Cool it to room temperature, make up the lost mass with acetonitrile, and filter it with a 0.45 μm microporous filter membrane to obtain the test solution.

[0112] (2) Same as Example 1, no further details will be given.

[0113] This comparative example separated and identified 81 chemical components in Xinnaoxin by UPLC-Q-TOF-MS, which indicates that acetonitrile has a poor extraction effect on water-soluble components and can identify fewer components.

[0114] Comparative Example 2

[0115] This comparative example provides a method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS, which is similar to Example 1, except that in step (2), the chromatographic column is replaced with a Waters acquity BEH amide chromatographic column (100 mm × 2.1 mm, 1.7 μm). The other conditions are the same as those in Example 1 and are not repeated here.

[0116] This comparative example used UPLC-Q-TOF-MS to separate and identify 76 chemical components in Xinnaoxin. This indicates that after changing the chromatographic column, the retention time was delayed and many peaks were not eluted.

[0117] Comparative Example 3

[0118] This comparative example provides a method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS, which is similar to Example 1, except that the elution procedure of the mobile phase and gradient elution in step (2) is different. The other conditions are the same as those in Example 1 and are not repeated here. The elution procedure of the mobile phase and gradient elution is as follows:

[0119] Mobile phase A was 25 mmol / L ammonium acetate in water, and mobile phase B was acetonitrile;

[0120] The elution program for gradient elution is as follows:

[0121] 0-2 min, 5% mobile phase A, 95% mobile phase B;

[0122] 2-18 min, 5% → 35% mobile phase A, 95% → 65% mobile phase B;

[0123] 18-20 min, 35% → 60% mobile phase A, 65% → 40% mobile phase B;

[0124] 20-22 min, 60% mobile phase A, 40% mobile phase B;

[0125] 22-22.1 min, 60% → 5% mobile phase A, 40% → 95% mobile phase B;

[0126] 22.1~30min, 5% mobile phase A, 95% mobile phase B.

[0127] In this comparative example, 71 chemical components in Xinnaoxin were separated and identified by UPLC-Q-TOF-MS method. The peaks were relatively concentrated, and many peaks were not eluted.

[0128] Comparative Example 4

[0129] This comparative example provides a method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS, which is similar to Example 1, except that in step (2), the mass-to-charge ratio scanning range is 200-2000 m / z, the ion source temperature (TEMP) is 500°C, the cluster fragmentation voltage (DP) is 100V, and the collision energy (CE) is 30eV. The other conditions are the same as those in Example 1 and will not be repeated here.

[0130] In this comparative example, 61 chemical components in Xinnaoxin were separated and identified by UPLC-Q-TOF-MS method, and no components with a mass-to-charge ratio less than 200 m / z were detected.

[0131] Compared with Comparative Examples 1 to 4, the method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS provided in Example 1 of the present invention identified 92 chemical components in Xinnaoxin, which is significantly more than the 61 to 81 chemical components in Comparative Examples 1 to 4. This shows that the method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS provided by the present invention can quickly, accurately and comprehensively analyze the chemical components of Xinnaoxin samples, providing a reference for promoting the secondary development of preparations based on Xinnaoxin and for further research and development of Xinnaoxin drugs.

[0132] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS, characterized in that: The steps include: (1) Preparation of test solution: Add the xinnaoxin sample into an alcohol solvent for extraction, filter, and obtain the test solution; (2) The test solution is tested using the UPLC-Q-TOF-MS method, wherein: The conditions for UHPLC include: The chromatographic column is a ZORBAX SB-C18 chromatographic column, the mobile phase A is a 0.1wt% to 0.5wt% formic acid aqueous solution, and the mobile phase B is methanol, and gradient elution is performed; The conditions for quadrupole time-of-flight tandem mass spectrometry include: Electrospray ion source, positive and negative ion modes, detection mode is information correlation acquisition mode; wherein, the mass-to-charge ratio scanning range is 100-1500 m / z, the ion source temperature is 520-580°C, the cluster fragmentation voltage is 75-85V, and the collision energy is 33-37eV.

2. The method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS according to claim 1, characterized in that: The specification of the ZORBAX SB-C18 chromatographic column is 3.0 mm×100 mm, and the filler diameter is 1.8 μm.

3. The method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS according to claim 1, characterized in that: The elution procedure of the gradient elution is as follows: 0-20min, 5%→20% mobile phase A, 95%→80% mobile phase B; 20-70 min, 20% → 60% mobile phase A, 80% → 40% mobile phase B; 70-75 min, 60% → 75% mobile phase A, 40% → 25% mobile phase B; 75-85min, 75%→90% mobile phase A, 25%→10% mobile phase B.

4. The method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS according to any one of claims 1 to 3, characterized in that: The flow rate of the gradient elution is 0.8-1.2 mL / min.

5. The method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS according to any one of claims 1 to 3, characterized in that: The column temperature of the chromatographic column is 25-30°C.

6. The method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS according to any one of claims 1 to 3, characterized in that: The conditions of the ultra high performance liquid chromatography also include: the injection volume is 8 to 12 μL.

7. The method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS as claimed in claim 1, characterized in that: The alcohol solvent is a methanol aqueous solution or anhydrous methanol with a mass concentration of not less than 50%.

8. The method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS according to claim 1 or 7, characterized in that, The mass volume ratio of the Xinnaoxin sample to the alcohol solvent is 1g:(17-23)mL.

9. The method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS as claimed in claim 1, characterized in that: The extraction adopts ultrasonic extraction; the frequency of the ultrasonic extraction is 38-42kHz, the power is 250-320W, and the extraction time is 25-35min.

10. The method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS according to claim 1, characterized in that: The conditions of the quadrupole time-of-flight tandem mass spectrometry also include: spray voltage is +(5300-5700)V, -(4300-4700)V; nebulizing gas pressure is 53-58psi; curtain gas pressure is 33-37psi; auxiliary gas pressure is 53-58psi; collision energy rolling range is 13-17eV.

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