Ultra-high performance liquid chromatography-mass spectrometry detection method for four-season antiviral mixture

Abstract

The invention provides an ultra-high performance liquid chromatography-quadrupole rod / electrostatic field orbitrap high-resolution mass spectrometry detection method for a four-season antiviral mixture, which adopts ultra-high performance liquid chromatography-quadrupole rod / electrostatic field orbitrap high-resolution mass spectrometry, and the optimal chromatographic conditions are as follows: 0-1.0 min, 98%-98% A and 2.0%-2.0% B; 1.0-14.0 min, 98%-70% of A and 2.0%-30.0% of B are added; in 14.0-25.0 min, 70%-0% of A is adopted, and 30%-100% of B is adopted; 25.0-28.0 min, 0%-0% of A and 100%-100% of B are included; from 28.0 min to 28.1 min, 0%-98% of A is adopted, and 100%-2% of B is adopted; 28.1-30.0 min, 98%-98% of A and 2%-2% of B are included; 148 chemical components, including 31 glycosides, 14 terpenoids, 54 flavonoids, 10 lignans, 23 organic acids, 3 alkaloids and 13 other types, are identified in total. The detection method can provide reference for internal quality detection of the traditional Chinese medicine, research on basic components of pharmacodynamic substances of the traditional Chinese medicine and authenticity identification of traditional Chinese medicine components.

Description

Technical Field

[0001] The invention belongs to the field of quality detection of chemical components of traditional Chinese medicines, and in particular relates to an ultra-high performance liquid phase-mass spectrometry detection method for a four-season antiviral mixture. Background Art

[0002] The Four Seasons Antiviral Mixture is a modified formula of Sangjuyin and Yinqiaosan in Treatise on Warm Diseases. It is used for upper respiratory tract infections, viral colds, influenza and other viral infections, with symptoms such as headache, fever, runny nose, cough, etc. The Shaanxi Province Traditional Chinese Medicine Treatment Plan for Pneumonia Infected by the New Coronavirus (Trial Second Edition), which was revised on January 31, 2020, includes the Four Seasons Antiviral Mixture in the New Coronavirus (2019-nCoV) infection patients who have symptoms such as wind-cold attacking the surface, qi deficiency and dampness stagnation during the clinical treatment period. It is recommended to use the Four Seasons Antiviral Mixture.

[0003] The applicant searched and sorted out the prior art documents related to the product and found that CN111830170A discloses a method for determining the content of components of the four-season antiviral mixture, using a KromasilC18 column, acetonitrile-0.085% phosphoric acid aqueous solution as the mobile phase, and eluting according to the following gradient: 0-30min, B98%→70%; 30-45min, B70%→55%; 45-60min, B55%→39%, detection wavelength: 277nm, 254nm; recording time: 60min; flow rate: 0.8ml·min-1; column temperature: 30℃; through ultra-fast liquid chromatography and gradient elution technology, the problems of insufficient precision and repeatability in the determination of the content of the four-season antiviral mixture are solved, and the accurate determination and stability assurance of the mixture components are achieved. Wang Siwang, Xiao Huimin, Liu Yang and others used UFLC to determine the mass concentrations of four components in Four Seasons Antiviral Mixture. The chromatographic column was a Kromasil C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase was acetonitrile -3.0 g·L -1 Phosphoric acid solution gradient elution; flow rate of 0.8 mL min-1; detection wavelength of 348 nm, column temperature of 35 °C. Determination of the mass concentrations of chlorogenic acid, 1,3-dicaffeoylquinic acid, luteolin and 3,5-dicaffeoylquinic acid in Four Seasons Antiviral Mixture. Wang Siwang, Xiao Huimin, He Yue, et al., Study on the ultrafast liquid chromatography fingerprint of Four Seasons Antiviral Mixture and determination of the contents of four components, 2015-02-15, discloses a method using UFLC method, chromatographic conditions: Kromasil C18 column (250 mm×4.6 mm, 5μm), mobile phase acetonitrile-0.6% phosphoric acid aqueous solution, gradient elution, flow rate of 0.8 ml min -1, with a detection wavelength of 260 nm and a column temperature of 30 °C, an ultra-fast liquid chromatography (UFLC) fingerprint of Siji Kangbingdu Mixture was established, and forsythoside A, phillyrin, liquiritin, and glycyrrhizic acid were determined.

[0004] However, there are also many defects and problems in the above detection methods, such as low detection sensitivity and relatively few detected components. However, in this study, an ultra-high performance liquid chromatography-quadrupole / electrostatic field orbitrap high-resolution mass spectrometry (UHPLC-Q Exactive Focus MS / MS) technology with high sensitivity, high resolution, and high throughput was used to rapidly separate and identify chemical components in traditional Chinese medicine compound preparations, providing a reference for the study of its effective substance basis, quality control, and mechanism of action. Summary of the Invention

[0005] The present invention provides an ultra-high performance liquid chromatography-mass spectrometry combined detection method for Siji Kangbingdu Mixture, using ultra-high performance liquid chromatography-quadrupole / electrostatic field orbitrap high-resolution mass spectrometry. This detection method has the advantages of short detection time, simple operation, and many detected components. A total of 148 chemical components were identified, including 31 glycosides, 14 terpenoids, 54 flavonoids, 10 lignans, 23 organic acids, 3 alkaloids, and 13 others. The above detection method can be used to provide a reference for the detection of the internal quality of the traditional Chinese medicine mixture of the present invention, the study of its effective substance basis components, and the identification of the authenticity of the components of the traditional Chinese medicine mixture.

[0006] The technical solution of this patent application of the present invention is as follows: An ultra-high performance liquid chromatography-mass spectrometry combined detection method for Siji Kangbingdu Mixture, the detection method comprising the following steps: (1) Preparation of the test solution: Weigh Siji Kangbingdu Mixture, place it in a centrifuge tube, add 70-80% methanol, and perform ultrasonic extraction. The ultrasonic frequency is 2800-3200 kHz, the ultrasonic power is 130-170 W, and the ultrasonic treatment time is 40-50 min. Place it in a centrifuge tube, with a centrifugation speed of 10000-14000 rpm and a centrifugation time of 5-15 min. Take the centrifuged solution to obtain it; ⑵ The chromatographic conditions are as follows: Ultra-high performance liquid chromatography using a Vanquish FlexUHPLC (Thermo Fisher Scientific, Inc., Waltham, MA, USA) equipped with an ACQUITY UPLC HSS T3 reversed-phase chromatographic column for chromatographic separation. The mobile phase consists of phase A being water-0.1% formic acid and phase B being acetonitrile; The elution ratio of the chromatographic mobile phase is as follows: from 0 to 1.0 min, 98% - 98% A, 2.0% - 2.0% B; from 1.0 to 14.0 min, 98% - 70% A, 2.0% - 30.0% B; from 14.0 to 25.0 min, 70% - 0% A, 30% - 100% B; from 25.0 to 28.0 min, 0% - 0% A, 100% - 100% B; from 28.0 to 28.1 min, 0% - 98% A, 100% - 2% B; from 28.1 to 30.0 min, 98% - 98% A, 2% - 2% B; the flow rate of the chromatographic mobile phase is 0.2 - 0.4 mL / min, and the column temperature of the chromatographic column is 35 - 45 °C; ⑶. Mass spectrometry conditions: A quadrupole orbitrap mass spectrometer equipped with a heated electrospray ionization source is used for mass spectrometry analysis. The ion source voltages for positive and negative ions are 3.5 - 4.0 kV and 3.0 - 4.0 kV respectively; the capillary heating temperature is 310 - 330 °C; the sheath gas pressure is 25 - 35 psi, and the auxiliary gas pressure is 8 - 12 psi; the solvent heating evaporation temperature is 290 - 310 °C; the collision gas is nitrogen, and the pressure is 1.2 - 1.7 mTorr; ⑷. Identification of chemical components Progenesis QI 3.0 software is used to process the collected mass spectrometry data. The steps are successively importing the original data, peak extraction, deconvolution, searching the reference database and the theoretical database, and performing multi-dimensional comprehensive judgment and analysis on the identification results through the retention time error of the reference, the mass error of the parent ion, the secondary fragment matching, the isotope distribution, the peak intensity, etc., to obtain the final results of the effective chemical components of traditional Chinese medicine.

[0007] Preferably, the concentration of methanol in step ⑴ of the detection method is 75%.

[0008] Preferably, the ultrasonic frequency in step ⑴ of the detection method is 3000 kHz, the ultrasonic power is 150 W, and the ultrasonic treatment time is 45 min.

[0009] Preferably, under the condition of a centrifugal speed of 12000 rpm in step ⑴ of the detection method, the centrifugation time is 10 min.

[0010] Preferably, the chromatographic column model in step ⑵ of the detection method is ACQUITY UPLC HSS T3, and the chromatographic column specification is: 100 mm × 2.1 mm, 1.8 μm.

[0011] Preferably, the flow rate of the chromatographic mobile phase in step ⑵ of the detection method is 0.3 mL / min, and the column temperature of the chromatographic column is 30 °C.

[0012] Preferably, in the mass spectrometry conditions of step (3) of the detection method, the ion source voltages of positive and negative ions are 3.7 kV and 3.5 kV respectively; the capillary heating temperature is 320 °C; the sheath gas pressure is 30 psi, and the auxiliary gas pressure is 10 psi; the solvent heating and evaporation temperature is 300 °C; both the sheath gas and the auxiliary gas are nitrogen; the collision gas is nitrogen, and the pressure is 1.5 mTorr.

[0013] Preferably, the detection method is used for the quality detection of Siji Kangbingdu Mixture, the study of effective substance components, and the application in the analysis and identification of traditional Chinese medicine compound components.

[0014] To further illustrate the creativity of the detection method of Siji Kangbingdu Mixture of the present invention, some experimental contents selected from the technical solution of the present invention are sorted out as follows.

[0015] Preparation of test solution After investigation, taking 200 μL of Siji Kangbingdu Mixture and ultrasonic extraction with 75% methanol for 45 min is the best preparation method.

[0016]

[0017] Mobile phase investigation In the table, the mobile phase and the elution gradient in the screening records are screened one by one interactively, and the optimal mobile phase is composed of phase A (water + 0.1% formic acid) and phase B (acetonitrile). The elution gradient table is as follows:

[0018] Due to the complex chemical components and significant polarity differences in Siji Kangbingdu Mixture, it is difficult to achieve effective separation and analysis solely relying on isocratic elution. Therefore, gradient elution is adopted to improve the peak shape and separation degree. In this application, acetonitrile (B) - 0.1% formic acid aqueous solution (A) is used for elution. In the previous experiments, only a small number of active ingredients in Siji Kangbingdu Mixture could be detected and identified. After 6 effective screenings in this application, the results of Screening Scheme 1 showed that the number of peaks was small, and the peaks were concentrated within 11 - 30 minutes without being separated. Therefore, in Screening 2, the mobile phase was changed and the elution time was shortened to 45 minutes, resulting in an increase in the number of peaks, but the total number was still not large enough. After changing the mobile phase from 100% - 70%, the separation peaks were bifurcated. In Screening 3, the ratio of 100% - 70% was deleted, and the peak of the active ingredient at 100% concentration was stably output. However, after the change, the retention time from 27 - 35 minutes was too long and almost no peaks appeared. In Screening 4, methanol was changed to acetonitrile, but using water for item A led to poor peak appearance and a small number of effective peaks. In Screening 5, using 0.1% phosphoric acid water for item A resulted in poor separation degree. Then, in Screening 6, item A was changed to 0.1% formic acid water. After changing the mobile phase, the number of peaks increased more, the peak shape became sharper, the separation degree was significantly improved, and the elution time was also shortened, greatly improving the efficiency. Finally, through a large number of experimental explorations and ratio adjustments, the optimal mobile phase ratio was determined as follows: 0 - 1 minute, B: 2%; 1 - 14 minutes, B: 2 - 30%; 14 - 25 minutes, B: 30 - 100%; 25 - 28 minutes, B: 100%; 28 - 28.1 minutes, B: 100% - 2%; 28.1 - 30 minutes, B: 2%. In this application, a quadrupole orbitrap mass spectrometer equipped with a heated electrospray ionization source (Q Exactive™, Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for mass spectrometry analysis. The ion source voltages for positive and negative ions were 3.7 kV and 3.5 kV respectively; compared with the total ion chromatogram (TIC) chromatogram, the positive and negative ion chromatograms had the advantages of low baseline, high abundance, significant signal, and more beautiful appearance.

[0019] The beneficial effects of the technical solution of this invention patent are as follows: ⑴. Through a large number of experimental explorations and attempts, the present invention finally found the optimal chromatographic conditions: from 0 to 1.0 min, 98% - 98% A, 2.0% - 2.0% B; from 1.0 to 14.0 min, 98% - 70% A, 2.0% - 30.0% B; from 14.0 to 25.0 min, 70% - 0% A, 30% - 100% B; from 25.0 to 28.0 min, 0% - 0% A, 100% - 100% B; from 28.0 to 28.1 min, 0% - 98% A, 100% - 2% B; from 28.1 to 30.0 min, 98% - 98% A, 2% - 2% B.

[0020] ⑵. The present invention uses ultra - performance liquid chromatography - hybrid quadrupole orbitrap high - resolution mass spectrometer (UHPLC - Q - Orbitrap HRMS) to detect the chemical components of traditional Chinese medicine in samples. By comparing the base - peak ion chromatograms of chemical components in positive and negative ion modes, it is found that the former has good responses in both positive and negative ion modes. To obtain the rich mass spectrometry information of sample chemical components to the greatest extent, two modes are used for injection, scanning and data acquisition respectively. By comparing with the reference database and theoretical database and combining manual verification, the components are qualitatively analyzed. There are 82 in the positive ion mode and 66 in the negative ion mode, and a total of 148 chemical components are screened out, including 9 of Houttuynia cordata, 33 of Platycodon grandiflorum, 9 of Morus alba leaves, 27 of Forsythia suspensa, 37 of Schizonepeta tenuifolia, 34 of Mentha haplocalyx, 15 of Perilla frutescens leaves, 11 of Armeniaca vulgaris, 7 of Phragmites australis roots, 38 of Chrysanthemum morifolium, and 43 of Glycyrrhiza uralensis. On the basis of obtaining more chemical components, more compound categories such as flavonoids and organic acids are also found, and alkaloid components such as magnoflorine and sophocarpine in the preparation are discovered for the first time.

[0021] ⑶. Compared with the previous research results of the chemical component analysis of Siji Kangbingdu in other existing technical literatures, the established determination method in this experiment shortens the detection time from 60 min to 30 min, greatly improves the detection efficiency, saves the use of mobile phase, and obtains more effective components with clear structures. The above - mentioned detection method can be used as a reference for the internal quality detection, the study of its pharmacodynamic material basis components, and the authenticity identification of traditional Chinese medicine components.

[0022] ⑷. For the first time, the present invention analyzed the cleavage pathways of 8 key pharmacodynamic components: forsythoside A, amygdalin, platycodin D, pulegone, chlorogenic acid, luteoloside, rutin, and aurantiamide acetate. These components include forsythoside A and chlorogenic acid, whose modern pharmacological effects are related to clearing heat and detoxifying of the mixture of the present invention. Amygdalin has a cough-relieving effect, thus providing a reference basis for the analysis of the effective substance components of the present invention. Description of the Drawings

[0023] Figure 1 , Chromatograms of the Four Seasons Anti-Virus Mixture under positive and negative ion modes.

[0024] Figure 2 , Chromatogram of the Four Seasons Anti-Virus Mixture under negative ion mode.

[0025] Figure 3 , Diagram of the secondary fragments and cleavage rules of amygdalin.

[0026] Figure 4 , Secondary fragments and cleavage rules of forsythoside A.

[0027] Figure 5 , Diagram of the secondary fragments and cleavage rules of platycodin D.

[0028] Figure 6 , Secondary fragments and cleavage rules of pulegone.

[0029] Figure 7 , Diagram of the secondary fragments and cleavage rules of chlorogenic acid.

[0030] Figure 8 , Secondary fragments and cleavage rules of rutin.

[0031] Figure 9 , Secondary fragments and cleavage rules of luteoloside.

[0032] Figure 10 , Secondary fragments and cleavage rules of aurantiamide acetate. Detailed Embodiments

[0033] Unless otherwise defined, the technical terms or scientific terms used in the specification and claims of this patent application for the present invention shall have the ordinary meanings understood by those of ordinary skill in the art to which the present invention pertains.

[0034] Example 1 Construction of the Ultra-High Performance Liquid Chromatography-Mass Spectrometry Detection Method for the Four Seasons Anti-Virus Mixture of the Present Invention 1 Instruments and Reagents 1.1 Instruments UltiMate 3000 ultra-high performance liquid chromatography system and Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, USA); BT25S electronic analytical balance (Sartorius Scientific Instruments < Beijing > Co., Ltd., accuracy 0.01 mg).

[0035] 2 Reagents Siji Kangbingdu mixture was provided by Shaanxi Haitian Pharmaceutical Co., Ltd., batch number: 2404071. Formic acid and acetonitrile were of mass spectrometry grade, methanol was of chromatographic grade, the rest of the reagents were of analytical grade, and water was ultrapure water.

[0036] 2 Methods and Results 1 Test Conditions Chromatographic conditions: Chromatographic separation was performed using a Vanquish Flex UHPLC (Thermo Fisher Scientific, Inc., Waltham, MA, USA) ultra-high performance liquid chromatography equipped with an ACQUITY UPLC HSS T3 (2.1 mm (inner diameter) × 100 mm (length), 1.8 μm (particle size)) (Waters Corp., MA, USA) reversed-phase chromatographic column. The mobile phase consisted of phase A (water + 0.1% formic acid) and phase B (acetonitrile). The elution gradient is shown in Table 3, and the flow rate was 0.3 mL / min. The column temperature was 40 °C. The injection volume was 6.0 μL.

[0037]

[0038] Mass spectrometry conditions: Mass spectrometry analysis was performed using a quadrupole orbitrap mass spectrometer (Q Exactive™, Thermo Fisher Scientific, Inc., Waltham, MA, USA) equipped with a heated electrospray ionization source. The ion source voltages for positive and negative ions were 3.7 kV and 3.5 kV, respectively; the capillary heating temperature was 320 °C; the sheath gas pressure was 30 psi, and the auxiliary gas pressure was 10 psi; the solvent heating and evaporation temperature was 300 °C; both the sheath gas and the auxiliary gas were nitrogen; the collision gas was nitrogen, and the pressure was 1.5 mTorr. Data was collected using the Full scan / dd-MS 2 mode. Full scan parameters: resolution 70000, automatic gain control target 1×10 6 , maximum isolation time 50 ms, mass-to-charge ratio scan range 100 – 1500; dd-MS 2 parameters: resolution 17500, automatic gain control target 1×10 5, maximum isolation time 50 ms, mass separation window 2, collision energies 10 V, 30 V, 60 V, intensity limit 1×10 5 .

[0039] 2.2 Sample Preparation Take 200 μL of the traditional Chinese medicine sample and place it in a 1.5 mL centrifuge tube. Add 600 μL of methanol and sonicate for 30 min. Place it in a 1.5 mL centrifuge tube and centrifuge at 4°C and 12000 rpm for 10 min. Take 100 μL of the solution and place it in an injection vial for measurement.

[0040] 2.3 Identification of Chemical Components Use Progenesis QI 3.0 software (Waters Corp., MA, USA) to process the collected mass spectrometry data. The steps are as follows: import the original data, peak extraction, and deconvolution. Search the reference database (TCM Pro 2.0) and the theoretical database (constructed through literature, public databases, etc.). Through the retention time error of the reference substance, the precursor ion mass error, the secondary fragment matching, the isotope distribution, the peak intensity, etc., conduct multi-dimensional comprehensive judgment and analysis on the identification results to obtain the final results. A total of 148 chemical components were resolved (66 of which were compared and confirmed with reference substances). Collect the chromatographic information in the positive and negative ion modes to obtain the corresponding base peak chromatogram (Base Peak Chromatogram, BPC). The chromatograms are shown in Figure 1 and Figure 2 . There are 82 chemical components in the positive ion mode and 66 in the negative ion mode, mainly from Houttuynia cordata, Platycodon grandiflorum, Morus alba, Forsythia suspensa, Schizonepeta tenuifolia, etc. Among the identified components, there are 31 glycosides, 14 terpenoids, 54 flavonoids, 10 lignans, 23 organic acids, 3 alkaloids, and 13 others. The specific information is shown in Table 4.

[0041]

[0042]

[0043] Glycosides: 31 chemical components. Taking amygdalin as an example, compound 30 was detected in positive ion mode with a retention time of 7.79 min, a quasi-molecular ion peak of m / z 475.1909 [M+NH4]+, and an inferred chemical formula of C20H27NO11. During pyrolysis, NH3 was removed to generate m / z 458.1691, C8H7NO was further removed to generate m / z 325.1127, C6H10O5 was further removed to generate m / z 163.0600, H2O was further removed to generate m / z 145.0496, and H2O was further removed to generate m / z 127.0394. By comparing with the reference substance, it was inferred that the compound was amygdalin. See the pyrolysis pathway for details. Figure 3 .

[0044] Taking forsythiaside A as an example, compound 67 was detected in positive ion mode with a retention time of 11.28 min and a quasi-molecular ion peak of m / z 609.1447 [M+H] + The chemical formula is estimated to be C 29 H 36 O 15 . C8H is removed during cleavage 10 O3 generates m / z 471.1487 and continues to remove C6H 10 O4 generates m / z 325.0916, and then removes C6H 10 O5 generated m / z 163.0388, and by comparing with the reference substance, it was inferred that the compound was Forsythoside A. The cleavage pathway is shown in Figure 4 .

[0045] Terpenes: 14 chemical components, platycodin, the compound was detected in negative ion mode, the retention time was 16.39min, the quasi-molecular ion peak was m / z 1223.5712 [MH]-, and the inferred chemical formula was C57H92O28. There are two pathways during cleavage. Pathway 1 removes C21H34O16 to generate m / z 681.3862, and path 2 removes C36H58O12 to generate m / z 541.1772, and then removes C5H8O4 to generate m / z 409.1354, and then removes C6H10O4 to generate m / z 263.0771. By comparing with the reference substance, it is inferred that the compound is platycodin D. See the cleavage pathway for details. Figure 5 .

[0046] Taking pulegone as an example, the compound was detected in the positive ion mode with a retention time of 20.15 min and a quasi-molecular ion peak of m / z 153.1273 [M+H] + , and the speculated chemical formula was C 10 H 16 O. During fragmentation, H2O was lost to form m / z 135.1168, followed by the loss of C2H2 to form m / z 109.1018, then the loss of H2 to form m / z 107.0862, and then the loss of CH2 to form m / z 93.0707, and finally the loss of C to form m / z 81.0709. By comparing with the reference substance, this compound was speculated to be pulegone. The fragmentation pathway is shown in Figure 6 .

[0047] Organic acids: There are 23 chemical components. Taking chlorogenic acid as an example, the compound was detected in the negative ion mode with a retention time of 7.17 min and a quasi-molecular ion peak of m / z 353.0875 [M-H] - , and the speculated chemical formula was C 16 H 18 O9. During fragmentation, C9H6O3 was lost to form m / z 191.0547. By comparing with the reference substance, this compound was speculated to be chlorogenic acid. The fragmentation pathway is shown in Figure 7 .

[0048] Flavonoids: There are 54 chemical components. Taking rutin as an example, the compound was detected in the negative ion mode with a retention time of 10.87 min and a quasi-molecular ion peak of m / z 609.1447 [M-H] - , and the speculated chemical formula was C 27 H 30 O 16 . During fragmentation, C 12 H 21 O9 was lost to form m / z 300.0275, and then C8H5O3 was lost to form m / z 151.0014. By comparing with the reference substance, this compound was speculated to be rutin. The fragmentation pathway is shown in Figure 8 .

[0049] Taking luteoloside compound 68 as an example, it was detected in the negative ion mode with a retention time of 11.38 min and a quasi-molecular ion peak of m / z 447.0935 [M-H] - , and the speculated chemical formula was C 21 H 20 O 11 . During fragmentation, C6H 10O5 generates m / z 285.0401, and continues to lose CO2 to generate m / z 241.0501. By comparing with the reference substance, it is speculated that this compound is Cynaroside. The fragmentation pathway is shown in Figure 9 .

[0050] Other categories: 13 chemical components. Taking asperglaucide as an example, compound 166 was detected in the positive ion mode, with a retention time of 20.5 min and a quasi-molecular ion peak of m / z 445.2083 [M+H] + , and the chemical formula is speculated to be C 27 H 28 N2O4. When fragmented, it loses C 11 H 15 NO2 to generate m / z 252.1021, continues to lose CO to generate m / z 224.1069, and continues to lose C8H9N to generate m / z 105.0342. By comparing with the reference substance, it is speculated that this compound is asperglaucide. The fragmentation pathway is shown in Figure 10 .

Claims

1. An ultra-high performance liquid chromatography-mass spectrometry detection method for Siji Kangbingdu Mixture, characterized in that, The detection method includes the following steps: ⑴ Preparation of the test solution: Weigh the Siji Kangbingdu mixture and place it in a centrifuge tube. Add 70 - 80% methanol and perform ultrasonic extraction. The ultrasonic frequency is 2800 - 3200 kHz, the ultrasonic power is 130 - 170 W, and the ultrasonic treatment time is 40 - 50 min. Place it in a centrifuge tube, centrifuge at a speed of 10000 - 14000 rpm for 5 - 15 min, and take the centrifuged solution to obtain the test solution. ⑵ Chromatographic conditions: Use a column equipped with an ultra - high - performance liquid chromatography column for chromatographic separation. The mobile phase consists of phase A (water - 0.1% formic acid) and phase B (acetonitrile). The elution ratio of the chromatographic mobile phase is as follows: 0 - 1.0 min, 98% - 98% A, 2.0% - 2.0% B; 1.0 - 14.0 min, 98% - 70% A, 2.0% - 30.0% B; 14.0 - 25.0 min, 70% - 0% A, 30% - 100% B; 25.0 - 28.0 min, 0% - 0% A, 100% - 100% B; 28.0 - 28.1 min, 0% - 98% A, 100% - 2% B; 28.1 - 30.0 min, 98% - 98% A, 2% - 2% B. The flow rate of the chromatographic mobile phase is 0.2 - 0.4 mL / min, and the column temperature is 35 - 45 °C. ⑶ Mass spectrometry conditions: Use a quadrupole orbitrap mass spectrometer equipped with a heated electrospray ionization source for mass spectrometry analysis. The ion source voltages for positive and negative ions are 3.5 - 4.0 kV and 3.0 - 4.0 kV respectively; the capillary heating temperature is 310 - 330 °C; the sheath gas pressure is 25 - 35 psi, and the auxiliary gas pressure is 8 - 12 psi; the solvent heating evaporation temperature is 290 - 310 °C; the collision gas is nitrogen, and the pressure is 1.2 - 1.7 mTorr. ⑷ Identification of chemical components Use Progenesis QI 3.0 software to process the collected mass spectrometry data. The steps are as follows: import the original data, peak extraction, deconvolution, search the reference database and theoretical database, and perform multi - dimensional comprehensive judgment and analysis on the identification results through the reference retention time error, precursor ion mass error, secondary fragment matching, isotope distribution, peak intensity, etc., to obtain the final results of the effective chemical components of traditional Chinese medicine.

2. The detection method according to claim 1, wherein The concentration of methanol in step ⑴ of the detection method is 75%.

3. The detection method according to claim 1, characterized in that, The ultrasonic frequency in step ⑴ of the detection method is 3000 kHz, the ultrasonic power is 150 W, and the ultrasonic treatment time is 45 min.

4. The detection method according to claim 1, wherein Under the condition of a centrifugal speed of 12000 rpm in step ⑴ of the detection method, the centrifugation time is 10 min.

5. The detection method according to claim 1, wherein The column model in step ⑵ of the detection method: ACQUITY UPLC HSS T3, and the column specification is: 100 mm × 2.1 mm, 1.8 μm.

6. The detection method according to claim 1, wherein The flow rate of the chromatographic mobile phase in step ⑵ of the detection method is 0.3 mL / min, and the column temperature is 30 °C.

7. The detection method according to claim 1, wherein In step (3) of the detection method, in the mass spectrometry conditions, the ion source voltages of positive and negative ions are 3.7 kV and 3.5 kV respectively; the capillary heating temperature is 320 °C; the sheath gas pressure is 30 psi, and the auxiliary gas pressure is 10 psi; the solvent heating and evaporation temperature is 300 °C; both the sheath gas and the auxiliary gas are nitrogen; the collision gas is nitrogen, and the pressure is 1.5 mTorr.

8. The detection method according to any one of claims 1 to 7, characterized in that, The said detection method is used for the quality detection of Siji Kangbingdu Mixture, the study of effective substance components, and the application in the analysis and identification of components of traditional Chinese medicine compound.

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