Method for detecting all 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 all 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 sample 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. According to the invention, 102 compounds in a heart and brain aoxin sample are finally identified, and the active ingredients of different types and sources have a treatment effect on a plurality of heart blood diseases in a synergistic or complementary manner. 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 full chemical components of xinnaoxin based on UPLC-Q-TOF-MS. Background Art

[0002] Xinnaoxin has the functions of invigorating qi and nourishing yin, promoting blood circulation and removing blood stasis. It is mainly used to treat dizziness, headache, palpitations, asthma, fatigue caused by qi and yin deficiency and blood stasis, as well as polycythemia caused by hypoxia. The main ingredients of Xinnaoxin include natural rhodiola rosea, wolfberry and fresh sea buckthorn syrup. Among them, the traditional Tibetan medicine rhodiola rosea is the main compound medicine, which has the functions of invigorating qi and promoting blood circulation, unblocking meridians and relieving asthma. It mainly includes chemical components such as salidroside, tyrosol and gallic acid; wolfberry has the functions of nourishing liver and kidney, improving essence and improving eyesight. Relevant studies have shown that organic acid components such as caffeic acid and p-coumaric acid in wolfberry are closely related to its functions; sea buckthorn has the functions of strengthening spleen and digestion, relieving cough and expectoration, promoting blood circulation and removing blood stasis. According to relevant studies, gallic acid and quercetin can be used as quality markers related to the efficacy of sea buckthorn. However, the current quality standards and related literature only detect the content of salidroside and betaine, which is difficult to use as an overall quality evaluation standard.

[0003] As a compound Chinese medicine composed of three Chinese medicines, Xinnaoxin contains complex chemical components. In the prior art, there are literature reports on a method of determining the content of seven components in Xinnaoxin by a one-test-multiple-evaluation method, which solves 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 simply, quickly, accurately and efficiently detecting the full chemical components in Xinnaoxin. Summary of the invention

[0004] In view of 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 includes the following steps:

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

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

[0009] The conditions for UHPLC include:

[0010] Positive ion mode: using a Waters acquity HSS T3 column, mobile phase A is 0.08 wt% to 0.12 wt% formic acid aqueous solution, mobile phase B is acetonitrile, and the first gradient elution is performed;

[0011] Negative ion mode: using a Waters acquity BEH amide column, mobile phase a was 22-28 mmol / L ammonium acetate aqueous solution, mobile phase b was acetonitrile, and the second gradient elution was performed;

[0012] The conditions for quadrupole time-of-flight tandem mass spectrometry include:

[0013] 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.

[0014] Compared with the prior art, the method for detecting the full chemical composition 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 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 102 compounds in the Xinnaoxin sample, and these compounds belong to flavonoids, anthraquinones, organic acids, phenylpropanoids and other compounds. The above active ingredients of different types and sources can have a therapeutic effect on many cardiovascular diseases in a synergistic or complementary manner.

[0015] The present invention adopts the UPLC-Q-TOF-MS method to quickly and accurately detect the various chemical components of the Xinnaoxin sample, providing a reference for promoting the secondary development of preparations based on Xinnaoxin and for further research and development of Xinnaoxin drugs.

[0016] Preferably, the specific steps of preparing the test solution include:

[0017] S1, taking a sample of Xinnaoxin, crushing and sieving it, adding an alcohol solvent to perform a primary extraction, and separating the solid from the liquid to obtain a filtrate;

[0018] S2, adding an alcohol solvent to the filtrate for secondary extraction, solid-liquid separation, and obtaining a test solution.

[0019] The invention adopts the method of adding alcohol solvent twice to extract the chemical components of the radix serrata, which can fully extract the target components and improve the extraction effect.

[0020] Preferably, the alcohol solvent is a methanol aqueous solution with a mass concentration of 70% to 80%.

[0021] Preferably, in step S1, the mass volume ratio of the xinnaoxin sample to the alcohol solvent is 1g:(45-55)mL.

[0022] Preferably, in step S2, the volume ratio of the filtrate to the alcohol solvent is 1:(0.8-1.2).

[0023] 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.

[0024] Preferably, the primary extraction and the secondary extraction are carried out by ultrasonic extraction, reflux extraction or vortex extraction, more preferably ultrasonic extraction.

[0025] Preferably, the frequency of the ultrasonic extraction is 38-42 kHz, the power is 280-320 W, and the time is 18-25 min.

[0026] The preferred ultrasonic extraction conditions of the present invention can further improve the extraction efficiency.

[0027] Preferably, the specification of the Waters acquity HSS T3 chromatographic column is 2.1 mm×100 mm, and the filler diameter is 1.8 μm.

[0028] Preferably, the specification of the Waters acquity BEH amide chromatographic column is 2.1 mm×100 mm, and the filler diameter is 1.7 μm.

[0029] Preferably, the elution procedure of the first gradient elution is as follows:

[0030] 0-1.5 min, 99% mobile phase A, 1% mobile phase B;

[0031] 1.5-15 min, 99% → 1% mobile phase A, 1% → 99% mobile phase B;

[0032] 15-20 min, 1% mobile phase A, 99% mobile phase B;

[0033] 20-21 min, 1% → 99% mobile phase A, 99% → 1% mobile phase B;

[0034] 21-30 min, 99% mobile phase A, 1% mobile phase B.

[0035] Preferably, the elution procedure of the second gradient elution is as follows:

[0036] 0-2min, 5% mobile phase a, 95% mobile phase b;

[0037] 2-18 min, 5% → 35% mobile phase a, 95% → 65% mobile phase b;

[0038] 18-20 min, 35%→60% mobile phase a, 65%→40% mobile phase b;

[0039] 20-22 min, 60% mobile phase a, 40% mobile phase b;

[0040] 22-22.1 min, 60% → 5% mobile phase a, 40% → 95% mobile phase b;

[0041] 22.1~30min, 5% mobile phase a, 95% mobile phase b.

[0042] Preferably, the flow rate is 0.18-0.22 mL / min and the column temperature is 25-30°C.

[0043] Preferably, the conditions of the ultra high performance liquid chromatography also include: an injection volume of 0.8 to 1.2 μL.

[0044] The preferred ultra-high performance liquid chromatography conditions of the present invention can more effectively separate the various chemical components of Xinnaoxin.

[0045] Preferably, the conditions of 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.

[0046] 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.

[0047] The present invention adopts the UPLC-Q-TOF-MS method, and realizes the rapid detection of multiple chemical components in Xinnaoxin by screening the conditions of liquid chromatography and mass spectrometry. The operation is simple, rapid, 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

[0048] Figure 1 This is the TIC diagram of the cerebral isin sample in the positive ion mode in Example 1 of the present invention;

[0049] Figure 2 This is the TIC diagram of the cerebral isotherm sample in the negative ion mode in Example 1 of the present invention;

[0050] Figure 3 is the MS graph of gallic acid (Compound 6) of the present invention;

[0051] Figure 4 is the MS pattern of ferulic acid (Compound 12) of the present invention;

[0052] Figure 5 is the MS pattern of chlorogenic acid (Compound 48) of the present invention;

[0053] Figure 6 is the MS graph of Taxifolin (Compound 1) of the present invention;

[0054] Figure 7 It is the MS chart of orientin (Compound 96) of the present invention. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solution 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 used to limit the present invention.

[0056] The instruments used in the embodiments of the present invention are shown in Table 1.

[0057] Table 1 Instruments used in the embodiments of the present invention

[0058] Serial number Instrument Name Manufacturer 1 1290 Infinity UPLC Agilent Technologies 2 Model 6545 Quadrupole Tandem Mass Spectrometer Agilent Technologies 3 Centrifuge Shanghai Anting Scientific Instrument Factory 4 JJ324BC electronic balance Changshu Shuangjie Testing Instrument Factory 5 Milli-Q Water Purifier Hebei Antu Trading Co., Ltd. 6 KQ-300DE CNC Ultrasonic Cleaner Kunshan Ultrasonic Instrument Co., Ltd.

[0059] 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 medicines: rhodiola rosea, wolfberry, and seabuckthorn pulp. The embodiment uses Xinnaoxin tablets (National Medicine Standard Z20090228) purchased from Donglutang Pharmaceutical Technology Co., Ltd.

[0060] Example 1

[0061] Example 1 provides a method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS, comprising the following steps:

[0062] (1) Take 1.0 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 50 mL volumetric flask, add 50 mL of 70 wt% methanol aqueous solution, perform ultrasonic extraction for 20 min, the ultrasonic frequency is 40 kHz, the power is 300 W, shake well and filter to obtain a filtrate; take 25 mL of the filtrate and put it into a 50 mL volumetric flask, add 25 mL of 80 wt% methanol aqueous solution, perform ultrasonic extraction for 20 min, the ultrasonic frequency is 40 kHz, the power is 300 W, shake well and filter to obtain the test solution.

[0063] (2) The test solution is tested using UPLC-Q-TOF-MS method, wherein:

[0064] I. The conditions of ultra-high liquid chromatography include:

[0065] ①Positive ion mode:

[0066] The chromatographic column was a Waters acquity HSS T3 column (100 mm × 2.1 mm, 1.8 μm);

[0067] Mobile phase A was 0.1 wt% formic acid in water, and mobile phase B was acetonitrile;

[0068] The flow rate was 0.2 mL / min;

[0069] The injection volume was 1 μL;

[0070] The column temperature was 30°C;

[0071] The first gradient elution, the elution program is as follows:

[0072] 0-1.5 min, 99% mobile phase A, 1% mobile phase B;

[0073] 1.5-15 min, 99% → 1% mobile phase A, 1% → 99% mobile phase B;

[0074] 15-20 min, 1% mobile phase A, 99% mobile phase B;

[0075] 20-21 min, 1% → 99% mobile phase A, 99% → 1% mobile phase B;

[0076] 21-30 min, 99% mobile phase A, 1% mobile phase B.

[0077] ②Negative ion mode:

[0078] The chromatographic column was a Waters acquity BEH amide column (100 mm × 2.1 mm, 1.7 μm);

[0079] Mobile phase a was 25 mmol / L ammonium acetate aqueous solution, and mobile phase b was acetonitrile;

[0080] The flow rate was 0.2 mL / min;

[0081] The injection volume was 1 μL;

[0082] The column temperature was 30°C;

[0083] The second gradient elution, the elution program is as follows:

[0084] 0-2min, 5% mobile phase a, 95% mobile phase b;

[0085] 2-18 min, 5% → 35% mobile phase a, 95% → 65% mobile phase b;

[0086] 18-20 min, 35%→60% mobile phase a, 65%→40% mobile phase b;

[0087] 20-22 min, 60% mobile phase a, 40% mobile phase b;

[0088] 22-22.1 min, 60% → 5% mobile phase a, 40% → 95% mobile phase b;

[0089] 22.1~30min, 5% mobile phase a, 95% mobile phase b.

[0090] II. The conditions for quadrupole time-of-flight tandem mass spectrometry include:

[0091] Electrospray ion source (ESI), positive ion mode, detection mode is information-dependent acquisition mode (IDA);

[0092] The mass-to-charge ratio scanning range was 100–1500 m / z;

[0093] The spray voltage (IS) is +5500V, -4500V;

[0094] The atomizing gas pressure (GS1) is 55 psi;

[0095] The air curtain pressure (CUR) is 35 psi;

[0096] Auxiliary gas pressure (GS2) is 55psi;

[0097] The ion source temperature (TEMP) was 550°C;

[0098] The cluster lysis voltage (DP) was 80 V;

[0099] The collision energy (CE) was 35 V;

[0100] The collision energy rolling range (CES) is 15V;

[0101] 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.

[0102] The total ion current chromatogram of Xinnaoxin sample is shown in Figures 1-2 , Figure 1 This is the TIC diagram of the Xinnaoxin sample in positive ion mode. Figure 2 This is the TIC diagram of the Xinnaoxin sample in negative ion mode. A total of 36 chemical substances were identified in the negative ion mode, and 66 chemical substances were identified in the positive ion mode.

[0103] Chemical composition analysis

[0104] Preparation of reference solution: Dissolve salidroside reference, quercetin reference, chlorogenic acid reference, chrysophanol reference, catechin reference and betaine reference in methanol, respectively, 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 reference substance solutions.

[0105] 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.

[0106] The above-mentioned reference solution was tested 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, and the peak position search of the target compound was realized by combining the HR-MS / MS Spectral Library1.1 database and the reference information, and the components corresponding to the target compound were screened out with accurate mass spectrometry (Mass error < 5ppm), and the secondary cleavage fragments were analyzed, thereby realizing accurate identification of the chemical components.

[0107] Example 1 102 chemical components in Xinnaoxin were separated and identified by UPLC-Q-TOF-MS method, of which 28 flavonoids were the main chemical components, and there were 19 organic acids, 7 phenylpropanoids, 10 anthraquinone compounds, cyanogenic glycosides, nucleosides and other compounds. The specific results are shown in Table 2.

[0108] Table 2 102 chemical components detected in Xinnaoxin based on UPLC-Q-TOF-MS method

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] Example 1 A total of 19 organic acid compounds were identified from Xinnaoxin, such as gallic acid, p-coumaric acid, quinic acid, etc. Organic acid compounds refer to a class of compounds containing carboxyl groups, phenolic hydroxyl groups, etc. in their structures. These compounds are often expressed as [MH] in mass spectrometry. - The ion peak appears and is easily formed in high-energy collisions [MH-H2O] - and [MH-CO2] - Neutral deletion fragment ions. The peak with a retention time of 8.875 (compound 6) is taken as an example to analyze the fragmentation rules.

[0118] The retention time of compound 6 is 8.875min, the quasi-ion peak in negative ion mode is m / z 169, the secondary fragment ion peak contains m / z 125, and m / z 169 forms fragment ions after removing -CO2. It can be seen that the compound has a molecular formula of C7H6O5, and in the case of 169 in actual measurement, the relative molecular weight of the peak is 169, which can determine that the compound is gallic acid. The MS fragmentation law of gallic acid is shown in formula 1, and the MS graph of gallic acid is shown in Figure 3 .

[0119]

[0120] Example 1 Seven phenylpropanoid compounds were identified from Xinnaoxin, such as p-coumarin-4-O-β-D-pyranoglucoside, 2,3-dihydroxybenzoic acid, ferulic acid, etc. Phenylpropanoid compounds refer to natural organic compounds containing one or more C3-C6 units in the basic parent nucleus. They are a variety of organic compounds synthesized by plants from amino acids phenylalanine and tyrosine. They can be divided into various chemical categories, such as simple phenylpropanol, coumarin, lignans, etc. Phenylpropanoid compounds usually contain multiple oxygen, hydroxyl, methoxyl and other groups connected to aromatic rings. Therefore, in their mass spectra, a series of fragment ion peaks of continuous loss of carbonyl, hydroxyl, water, methyl, methoxyl and other groups often appear. The peaks with retention times of 1.708 and 3.57 (compounds 12 and 48) are taken as examples for the analysis of the cleavage rules.

[0121] The retention time of compound 12 is 1.708 min, and the quasi-ion peak in negative ion mode is m / z 193. After removing one molecule of -CH3, m / z 178 is formed, and then one molecule of -CO2 is removed to form m / z 134. It can be seen that the molecular formula of the compound is C 10 H 10 O4, relative molecular weight is 194; the relative molecular weight of the peak actually measured is 193, and the theoretical relative molecular weight is 193, which can identify the compound as ferulic acid. The MS fragmentation law of ferulic acid is shown in Formula 2, and the MS graph of ferulic acid is shown in Figure 4 .

[0122]

[0123] The retention time of compound 48 is 3.57min, and the quasi-ion peak in positive ion mode is m / z 353. m / z191, 179, 173, and 135 are fragments produced by its cleavage. It is speculated that the cleavage pattern is that m / z191 and m / z 179 are first cleaved to form m / z 191 and m / z 179, and then m / z 191 loses a molecule of H2O to generate m / z 173, and m / z 179 loses a molecule of CO2 to form m / z 135. It can be judged that the compound is chlorogenic acid. The MS cleavage pattern of chlorogenic acid is shown in formula 3, and the MS graph of chlorogenic acid is shown in Figure 5 .

[0124]

[0125] Example 1 Ten anthraquinone compounds were isolated from Xinnaoxin, such as rhubarb, p-phenolic hydroxyl-β-D-pyrrolidine glucoside and clematis. Anthraquinone compounds refer to chemical components with quinone structures. 9,10-anthraquinone is the most common type of natural anthraquinone. Its entire molecule constitutes a conjugated system. C9, C 10It is at the highest oxidation level and is relatively stable. The parent nucleus of anthraquinone is usually substituted by hydroxyl, methyl, carboxyl, etc.

[0126] Example 1 28 flavonoids were identified from the Xinnaoxin solution, such as taxifolin, afudamidin, salidroside, quercetin, eriodictyol, salidroside, luteolin, etc. Flavonoids refer to a class of compounds whose basic parent nucleus is 2-phenylchromone, which is ubiquitous and abundant in many substances, and is also the most abundant class in Xinnaoxin. Flavonoids have strong mass spectrometric characteristics, and generally lose methyl, carboxyl and other groups to form fragment ion peaks. The peaks with retention times of 3.008 and 8.735 (compounds 1 and 96) are taken as examples for analysis of the cleavage rules.

[0127] The retention time of compound 1 is 3.008min, and the quasi-ion peak in the positive ion mode is m / z 305, which is easy to lose -C8H8O3 to form a fragment ion of m / z 153, and then the ion loses two O to form m / z 123; at the same time, the C ring of m / z 305 is easy to lose a molecule of -H2O to form a fragment ion of m / z 287, which further breaks down and loses two molecules of CO to form m / z 259 and m / z 231 respectively, and m / z 259 loses the B ring and rearranges to form m / z 121 and m / z 149. The compound has a molecular formula of C 15 H 12 The molecular formula of O7 and the relative molecular weight of 305; the theoretical calculated value of the relative molecular weight is 305.06, and the actual measured value is 305.05, which can determine that the compound is taxifolin. The MS fragmentation law of taxifolin is shown in Formula 4, and the MS graph of taxifolin is shown in Figure 6 .

[0128]

[0129] The retention time of compound 96 is 8.735min, and the quasi-molecular ion peak in the positive ion mode is m / z449, of which m / z 431.2, 413.2, 395.2, 383.2, 366.3, 352.3, 329.3, and 299.2 are fragments generated by its cleavage. It is speculated that the cleavage rule is to first detach from a molecule of -H2O to form a fragment ion m / z431, and then detach from a molecule of -H2O to generate m / z 413. Fragment m / z 395 cleaves to m / z 367 by detaching from a molecule of -CO. It can be determined that the compound is orientin, and the specific cleavage rule is shown in Formula 5. The MS graph of orientin is shown in Figure 7 .

[0130]

[0131] Example 1 also identified 15 compounds, including cyanogenic glycosides, phenylpropyl esters, sugars, phenylacetones, volatile oils, sterols, esters, coumarins, phenols, glycosides, phenylethanol, steroids, polyols, saponins, and indole derivatives.

[0132] 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, 102 chemical components in the Xinnaoxin sample can be identified, but the peak shape is slightly extended or shifted, and the peak response height is slightly different.

[0133] Comparative Example 1

[0134] 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:

[0135] (1) Take 1.0 g of the 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 50 mL volumetric flask, add 50 mL of acetonitrile and perform ultrasonic extraction for 20 min. The ultrasonic frequency is 40 kHz and the power is 300 W. After shaking, filter and obtain the filtrate; take 25 mL of the filtrate and put it into a 50 mL volumetric flask, add 25 mL of acetonitrile (diluted to 10 mg / mL) and perform ultrasonic extraction for 20 min. The ultrasonic frequency is 40 kHz and the power is 300 W. After shaking, filter and obtain the test solution.

[0136] (2) Same as Example 1.

[0137] This comparative example separated and identified 82 chemical components in Xinnaoxin by UPLC-Q-TOF-MS method. This shows that acetonitrile has a poor extraction effect on water-soluble components, few components can be identified, and the peaks are relatively concentrated, with poor separation.

[0138] Comparative Example 2

[0139] 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 in positive ion mode is replaced with a WondaSil C18-WR chromatographic column (4.6 mm×250 mm, 5 μm), and the other conditions are the same as those in Example 1.

[0140] This comparative example separated and identified 77 chemical components in Xinnaoxin by UPLC-Q-TOF-MS method. This shows that after changing the chromatographic column, the retention time is delayed, many peaks are not eluted, and the peak intensity is low, and the separation is poor.

[0141] Comparative Example 3

[0142] 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 mobile phase and gradient elution procedure of the negative ion mode in step (2) are different, and the other conditions are the same as Example 1. The elution procedure of the mobile phase and gradient elution is as follows:

[0143] Mobile phase a is 0.1 wt% formic acid aqueous solution, and mobile phase b is acetonitrile;

[0144] Gradient elution, the elution program is as follows:

[0145] 0-10 min, 95% mobile phase A, 5% mobile phase B;

[0146] 10-38 min, 95% → 0% mobile phase A, 5% → 100% mobile phase B;

[0147] 38-40 min, 0% mobile phase A, 100% mobile phase B.

[0148] In this comparative example, 73 chemical components in Xinnaoxin were separated and identified by UPLC-Q-TOF-MS method. The peaks were relatively concentrated, the peak intensity was low, and the separation degree was poor.

[0149] Comparative Example 4

[0150] 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 800-2000 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.

[0151] In this comparative example, 55 chemical components in Xinnaoxin were separated and identified by UPLC-Q-TOF-MS method, and no components with mass-to-charge ratio less than 200 m / z were detected.

[0152] Compared with Comparative Examples 1 to 4, the method for detecting the full chemical components of Xinnaoxin based on UPLC-Q-TOF-MS provided in the embodiment of the present invention identified 102 chemical components in Xinnaoxin, which is significantly more than the 55 to 82 chemical components in Comparative Examples 1 to 4. This shows that the method for detecting the full 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, and provide a reference for promoting the secondary development of preparations based on Xinnaoxin and for more in-depth research and development of Xinnaoxin drugs.

[0153] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope 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 UPLC-Q-TOF-MS method, wherein: The conditions for UHPLC include: Positive ion mode: using a Waters acquity HSS T3 column, mobile phase A is 0.08 wt% to 0.12 wt% formic acid aqueous solution, mobile phase B is acetonitrile, and the first gradient elution is performed; Negative ion mode: using a Waters acquity BEH amide column, mobile phase a was 22-28 mmol / L ammonium acetate aqueous solution, mobile phase b was acetonitrile, and the second gradient elution was 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 specific preparation steps of the test solution include: S1, taking a sample of Xinnaoxin, crushing and sieving it, adding an alcohol solvent to perform a primary extraction, and separating the solid from the liquid to obtain a filtrate; S2, adding an alcohol solvent to the filtrate for secondary extraction, solid-liquid separation, and obtaining a test solution.

3. The method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS as claimed in claim 2, characterized in that: The alcohol solvent is a methanol aqueous solution with a mass concentration of 70% to 80%; and / or The first extraction and the second extraction are ultrasonic extraction; the frequency of the ultrasonic extraction is 38-42 kHz, the power is 280-320 W, and the time is 18-25 min.

4. The method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS as claimed in claim 2, characterized in that: In step S1, the mass volume ratio of the xinnaoxin sample to the alcohol solvent is 1 g: (45-55) mL; and / or In step S2, the volume ratio of the filtrate to the alcohol solvent is 1:(0.8-1.2).

5. 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 Waters acquity HSS T3 chromatographic column is 2.1 mm×100 mm, and the filler diameter is 1.8 μm; and / or The specification of the Waters acquity BEH amide chromatographic column is 2.1 mm×100 mm, and the filler diameter is 1.7 μm.

6. 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 first gradient elution is as follows: 0-1.5 min, 99% mobile phase A, 1% mobile phase B; 1.5-15 min, 99% → 1% mobile phase A, 1% → 99% mobile phase B; 15-20 min, 1% mobile phase A, 99% mobile phase B; 20-21 min, 1% → 99% mobile phase A, 99% → 1% mobile phase B; 21-30 min, 99% mobile phase A, 1% mobile phase B.

7. The method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS according to claim 1, characterized in that: The elution program of the second gradient elution is as follows: 0-2min, 5% mobile phase a, 95% mobile phase b; 2-18 min, 5% → 35% mobile phase a, 95% → 65% mobile phase b; 18-20 min, 35%→60% mobile phase a, 65%→40% mobile phase b; 20-22 min, 60% mobile phase a, 40% mobile phase b; 22-22.1 min, 60% → 5% mobile phase a, 40% → 95% mobile phase b; 22.1~30min, 5% mobile phase a, 95% mobile phase b.

8. The method for detecting the chemical components of Xinnaoxin based on UPLC-Q-TOF-MS according to claim 6 or 7, characterized in that: The flow rate is 0.18-0.22 mL / min, and the column temperature is 25-30°C.

9. 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 ultra high performance liquid chromatography also include: injection volume 0.8 to 1.2 μL.

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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