Qualitative and quantitative method for detecting active ingredients of root-strengthening and cough-relieving granules by UPLC-MS / MS (ultra performance liquid chromatography-mass spectrometry / mass spectrometry)

Abstract

The invention relates to a qualitative and quantitative method for detecting active ingredients of root-strengthening and cough-relieving granules through UPLC-MS / MS, and belongs to the technical field of medicine analysis and detection. The four active ingredients of liquiritigenin, isoliquiritigenin, luteolin and bakuchiol in a sample can be detected at the same time. The method comprises the following steps: pretreating a sample by a 70% methanol ultrasonic extraction method to obtain a target sample solution, and detecting by using an SCIEX ExionLC AE high-pressure liquid phase system and an AB SCIEX 4500 mass spectrometer in combination with an MRM mode. Carrying out gradient elution on a liquid phase by using an HSS T3 C18 chromatographic column and 0.1% formic acid water-acetonitrile as a mobile phase; the mass spectrum is electrospray negative ion scanning, and a specific ion source and MRM parameters are set. The method can rapidly separate the four components, is short in detection period, high in sensitivity and strong in matrix interference resistance, can be accurately qualitative and quantitative, and provides support for preparation quality evaluation, pharmacodynamic mechanism research and clinical reasonable medication.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis and detection technology, and relates to a qualitative and quantitative method for detecting the active ingredients of Gubenzhike granules by UPLC-MS / MS. Background Technology

[0002] Gubenzhike Granules are a traditional Chinese medicine compound preparation composed of Ophiopogon japonicus, Schisandra chinensis, roasted licorice root, and Psoralea corylifolia. It has the effects of invigorating qi and consolidating the exterior, strengthening the spleen and kidneys, relieving cough and asthma. Clinically, it is used to treat cough caused by spleen deficiency and phlegm accumulation, and kidney qi deficiency, as well as chronic bronchitis, emphysema, bronchial asthma and other diseases.

[0003] In the quality control and efficacy mechanism research of Gubenzhike granules, accurate analysis of active ingredients is crucial. However, existing technologies have the following shortcomings: Limitations of traditional detection methods: Traditional analytical techniques such as high performance liquid chromatography have significant shortcomings in detection sensitivity, separation efficiency, and simultaneous quantification of multiple components, making it difficult to meet the detection needs of trace active ingredients in complex traditional Chinese medicine compound prescriptions.

[0004] Severe matrix interference: The complex composition of traditional Chinese medicine compound formulas and the large number of coexisting impurities in the matrix can easily interfere with the detection of target active ingredients, resulting in insufficient ability of existing methods to distinguish between target ingredients and impurities, and making it difficult to guarantee the accuracy of content determination results.

[0005] Lack of proprietary methods: There are currently no reports on systematic quantitative analysis methods for the four key antitussive active ingredients in Gubenzhike granules, namely glycyrrhizin, isoglycyrrhizin, luteolin and psoralen, which directly restricts the improvement of the basic research on the efficacy components and the quality evaluation system of this preparation.

[0006] In recent years, ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC-MS / MS) has become an important tool for the analysis of trace components in complex matrices due to its advantages of high resolution, high sensitivity, and multiple reaction monitoring (MRM) mode. However, how to optimize chromatographic separation conditions and mass spectrometry parameters for specific components of Gubenzhike granules, and establish a dedicated UPLC-MS / MS detection method to achieve simultaneous and accurate quantification of four active ingredients, remains a technical challenge that urgently needs to be solved.

[0007] Therefore, there is an urgent need to develop a comprehensive and systematic quantitative analytical method for the key active ingredients in Gubenzhike granules, so as to provide a scientific basis for the quality evaluation, pharmacodynamic mechanism research and rational clinical use of this preparation. Summary of the Invention

[0008] The purpose of this invention is to provide a qualitative and quantitative method for detecting the active ingredients of Gubenzhike granules by UPLC-MS / MS. This method can achieve rapid separation, accurate qualitative and quantitative analysis of four active ingredients (glycyrrhizin, isoglycyrrhizin, luteolin and psoralen) in the same analytical system. It features high detection sensitivity, excellent separation efficiency, strong resistance to matrix interference, and short detection cycle.

[0009] The objective of this invention can be achieved through the following technical solutions: A qualitative and quantitative method for simultaneous detection of four active ingredients in a Gubenzhike granule sample by UPLC-MS / MS, wherein the four active ingredients are glycyrrhizin, isoglycyrrhizin, luteolin, and psoralen; the method includes the following steps: X1. Pre-treat the sample of Gubenzhike Granules to be tested to obtain the target sample solution; X2. The target sample solution was detected by ultra-high performance liquid chromatography in tandem triple quadrupole mass spectrometry, combined with multiple reaction monitoring (MRM) mode, to achieve qualitative identification and content determination of four active ingredients; The sample pretreatment method was 70% methanol ultrasonic extraction, the liquid chromatography system used was a SCIEX ExionLC AE high-performance liquid chromatography system, and the mass spectrometer was an AB SCIEX 4500.

[0010] As a preferred embodiment of the present invention, the detection conditions for the ultra-high performance liquid chromatography are as follows: Chromatographic column: A C18 column is selected, preferably the HSS T3 model, with a column length of 100 mm, an inner diameter of 2.1 mm, a particle size of 1.8 μm, and a column temperature of 35℃; Mobile phase: Phase A is 0.1% formic acid solution, and Phase B is acetonitrile; Gradient elution conditions: At 0.00 min, phase A accounts for 70% and phase B accounts for 30%, with a flow rate of 0.3 mL / min; at 3.00 min, the phase A ratio is adjusted to 5% and the phase B ratio is adjusted to 95%, with the flow rate still at 0.3 mL / min; at 5.00 min, the ratio of phase A to phase B is maintained at 5% and the flow rate at 95%, with a flow rate of 0.3 mL / min; at 5.01 min, the initial ratio of phase A to phase B is restored to 70% and the flow rate at 30%, with a flow rate maintained at 0.3 mL / min; at 7.00 min, the ratio of phase A to phase B is maintained at 70% and the flow rate at 30%, with a flow rate of 0.3 mL / min. Injection volume: 5 μL; Sample diluent: 70% methanol-water.

[0011] As a preferred embodiment of the present invention, the pretreatment step of the sample to be tested specifically includes: W1. Accurately weigh 0.02g of the Gubenzhike Granules sample and place it in a centrifuge tube; W2. Add 10 mL of 70% methanol-water to the centrifuge tube, vortex to mix, and then perform ultrasonic extraction at room temperature; W3. After ultrasonic extraction, replenish the weight of the extraction reagent lost due to ultrasonic evaporation with 70% methanol-water solution. W4. Centrifuge the above solution at 4200 rpm for 10 min at room temperature and collect the supernatant; W5. Filter the collected supernatant through a 0.22μm organic nylon filter membrane, preferably a 0.22μm organic nylon filter membrane from the Jinteng brand; W6. Dilute the filtered supernatant with 70% methanol-water by 4 times to obtain the target sample solution, ready for instrument detection.

[0012] As a preferred embodiment of the present invention, the conditions for ultrasonic extraction are: ultrasonic power 300W, ultrasonic frequency 40kHz, and ultrasonic time 30min.

[0013] As a preferred embodiment of the present invention, the detection conditions for the triple quadrupole mass spectrometer are as follows: Ion source: Electrospray ionization source, scanning mode is negative ion scanning; Ion source parameters: ionization voltage is -4500V, ion source temperature is 550℃, atomizing gas pressure is 50psi, auxiliary heating gas pressure is 55psi, curtain gas pressure is 35psi, collision gas / collision activated dissociation intensity is Medium-9. Multiple reaction monitoring (MRM) conditions: The precursor ion of glycyrrhizin was 255.080, with corresponding daughter ions of 119.010 and 134.930, both with a residence time of 50 ms. The collision voltage and declustering voltage for daughter ion 119.010 were -45.0 V and -70.0 V, respectively, while those for daughter ion 134.930 were -22.0 V and -65.0 V. The precursor ion of isoglycyrrhizin was 255.010, with corresponding daughter ions of 119.010 and 134.940, both with a residence time of 50 ms. The collision voltage and declustering voltage for daughter ion 119.010 were -31.0 V and -50 V, respectively, while those for daughter ion 134.940 were -22.0 V and -50 V. Luteolin... The parent ion is 285.08, and the corresponding daughter ions are 132.970 and 150.930, respectively. The residence time for both is 50 ms. The collision voltage and declustering voltage of daughter ion 132.970 are -48V and -65V, respectively, while those for daughter ion 150.930 are -36V and -65V, respectively. The parent ion for psoralen is 255.180, and the corresponding daughter ion is 172.000. The residence time is 50 ms, and the collision voltage is -25V and the declustering voltage is -60V. Among the above compounds, the daughter ions labeled for quantitative detection are 134.930 for glycyrrhizin, 119.010 for isoglycyrrhizin, 132.970 for luteolin, and 172.000 for psoralen. The remaining daughter ions are used for qualitative detection.

[0014] As a preferred technical solution of the present invention, the determination criteria for qualitative detection are as follows: the retention time of each active ingredient in the target sample solution deviates from the retention time of the standard within ±0.1 min, and the corresponding qualitative ion pair response signal intensity meets the detection requirements; wherein, the retention time of glycyrrhizin is 2.1 min, the retention time of isoglycyrrhizin is 2.6 min, the retention time of luteolin is 2.1 min, and the retention time of psoralen is 4.5 min.

[0015] As a preferred technical solution of the present invention, the quantitative detection calculation method is as follows: First, prepare a mixed standard solution of four active ingredients with six different concentration gradients, and detect them under the chromatographic and mass spectrometric conditions described in claims 2 and 5. Plot a standard curve with the standard solution concentration as the abscissa and the corresponding quantitative ion pair peak area as the ordinate, and obtain a linear regression equation. Then, substitute the quantitative ion pair peak area of ​​the target sample solution into the linear regression equation, and calculate the content of the four active ingredients in the Gubenzhike granules sample by combining the sample weight, extraction volume and dilution factor.

[0016] As a preferred embodiment of the present invention, the concentration gradient of the mixed standard solution is as follows: Glycyrrhizin: 0.15 ng / mL, 0.3 ng / mL, 1.5 ng / mL, 7.5 ng / mL, 15 ng / mL, 30 ng / mL; Isoliquiritigenin: 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL; Luteolin: 0.1 ng / mL, 0.2 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL; Bakuchiol: 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL; Furthermore, the correlation coefficients of the standard curves for the four active ingredients were all greater than 0.99, indicating a good linear relationship.

[0017] The beneficial effects of this invention are: (1) Based on UPLC-MS / MS technology combined with MRM mode, this invention matches exclusive chromatographic columns and mass spectrometry parameters to improve the detection sensitivity of four active ingredients to the level of 0.1 ng / mL; at the same time, through mass spectrometry characteristic ion pair screening, the interference of complex matrix of traditional Chinese medicine compound is effectively eliminated, ensuring accurate identification and quantification of target ingredients, and solving the problem that traditional methods are difficult to distinguish between target ingredients and impurities.

[0018] (2) By optimizing the gradient elution conditions, the rapid separation of four active ingredients in the same analytical system was achieved, and the single detection cycle was controlled within 7 minutes, which greatly shortened the detection time. Moreover, there was no need to establish separate detection methods for the four ingredients, which significantly improved the detection throughput and is suitable for the analysis of batch samples.

[0019] (3) The methodological evaluation results showed that the spiked recoveries of the four active ingredients were between 80% and 120%, the relative standard deviation (RSD) was less than 15%, and the linear correlation coefficients (r²) were all greater than 0.99, indicating that the method has good accuracy, repeatability and linearity, and can be used for the stable detection of active ingredients in Gubenzhike granules.

[0020] (4) This invention is the first to achieve systematic quantitative analysis of four key antitussive active ingredients in Gubenzhike Granules: glycyrrhizin, isoglycyrrhizin, luteolin and psoralen. The accurate content data of the four ingredients were obtained (average contents of 45.64 μg / g, 6.73 μg / g, 0.25 μg / g and 98.55 μg / g, respectively). This provides key technical support for elucidating the antitussive mechanism of the preparation, improving the quality evaluation system and guiding rational drug use in clinical practice. Attached Figure Description

[0021] Figure 1The peak chromatogram of a mixed standard solution of glycyrrhizin, isoglycyrrhizin, luteolin and psoralen is shown at -5. Figure 2 Peak spectrum of blank solution Figure 3 Peak spectra of four compounds in Gubenzhike granules Detailed Implementation To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0022] Example 1 Establishment of UPLC-MS / MS methods for glycyrrhizin, isoliquiritigenin, luteolin and psoralen 1.1 Experimental Apparatus: Mass spectrometry system: AB SCIEX 4500. Liquid chromatography system: SCIEX ExionLC AE high-performance liquid chromatography system. Chromatographic column: Waters ACQUITY HSS T3 column, 100 mm in length, 2.1 mm in inner diameter, and 1.8 μm in particle size.

[0023] 1.2 Preparation of standard products: Glycyrrhizin, isoliquiritigenin, luteolin and psoralen stock solutions of 20 μg / mL were diluted with 50% methanol and water to a concentration of 50 ng / mL. The single standard solutions were directly injected into the mass spectrometry system using a syringe to explore and optimize the parent ion, daughter ion, collision energy (CE) and declustering voltage (DP) of the four active ingredients.

[0024] 1.3 Establishment and optimization of mass spectrometry parameters: Mass spectrometry detection conditions for the four active ingredients: The ion source was an electrospray ionization source (ESI), and the ion source parameters were as follows: ionization voltage: -4500 V; ion source temperature: 550℃; nebulizing gas (GS1): 50 psi; auxiliary heating gas (GS2): 55 psi; curtain gas (CUR): 35 psi; collision gas / collision activated dissociation (CAD): Medium-9; scanning mode: negative ion scanning, and the multiple reaction monitoring (MRM) conditions are shown in Table 1.

[0025] Table 1. Multiple reaction monitoring conditions for glycyrrhizin, isoliquiritigenin, luteolin, and psoralen. Note: 'a' is used for quantitative detection, and the other ion pair is used for qualitative detection.

[0026] 1.4 Establishment and optimization of liquid phase conditions: A C18 column was used, with a column temperature of 35℃; the mobile phase A was 0.1% formic acid in water, and the mobile phase B was acetonitrile; the gradient elution conditions are shown in Table 2 below. Table 2 Liquid phase gradient elution ratio The C18 chromatographic column is model HSS T3, with a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm.

[0027] The standard diluent was 70% methanol-water, the flow rate was 0.3 mL / min, and the injection volume was 5 μL.

[0028] 1.5 Establishment of a sample pretreatment method for Gubenzhike granules: Accurately weigh the sample of Gubenzhike granules, add 10 mL of 70% methanol-water mixture, vortex to mix, and extract by sonication at room temperature. Make up the weight of the lost extraction reagent with 70% methanol-water mixture. Centrifuge at 4200 rpm for 10 min at room temperature, carefully collect the supernatant, filter the supernatant through a 0.22 μm organic nylon filter membrane, dilute it, and then analyze it.

[0029] Weigh 0.02 g of the sample to ensure that the sample is fully extracted in the limited extraction solution.

[0030] The ultrasonic conditions were 30 min (300 W, 40 kHz). The extraction solution was diluted 4 times before being analyzed to eliminate the influence of the matrix.

[0031] The organic nylon filter membrane used was the Jinteng brand 0.22 μm organic nylon filter membrane. The recovery rates of different brands of organic nylon filter membranes are shown in Table 3 below. The test was conducted using a 50 ng / mL mixed standard solution.

[0032] Table 3. Effect of different brands of filter membranes on the recovery rate of the analyte compounds As shown in the table above, except for the Jinteng organic nylon filter membrane, which has a recovery rate of 85-115% for glycyrrhizin, isoliquiritigenin, luteolin and psoralen, the other filter membranes all have some adsorption of analytes and insufficient recovery, especially the two active ingredients, isoliquiritigenin and luteolin, which have serious losses.

[0033] Example 2 Methodological evaluation 2.1 Specificity Analysis: To investigate the specificity of the mass spectrometry method for the four active substances, a mixed standard solution of the four compounds was prepared for precise injection. The peak spectra of the mixed standard solution of glycyrrhizin, isoglycyrrhizin, luteolin, and psoralen in the developed liquid chromatography and mass spectrometry methods are shown below. Figure 1As shown, the peak spectrum of the blank solvent is as follows: Figure 2 As shown, the elution times for glycyrrhizin ion pair were 2.1 min, isoglycyrrhizin ion pair 2.6 min, luteolin ion pair 2.1 min, and psoralen ion pair 4.5 min. Although glycyrrhizin and isoglycyrrhizin ion pairs were identical, their elution times differed, allowing for differentiation and quantification based on elution times. Luteolin and glycyrrhizin had identical elution times, but their ion pairs differed, thus allowing for identification based on ion pair responses. Therefore, the established mass spectrometry and liquid chromatography method was applied to the analysis of these four components, demonstrating good separation, favorable peak shapes, absence of solvent peak interference, and good specificity.

[0034] 2.2 Establishment of Standard Curves and Analysis of Linear Relationships To further evaluate the quantitative analytical capability of the method, 20 μg / mL of glycyrrhizin, isoglycyrrhizin, luteolin, and psoralen stock solutions were used to prepare mixed standard solutions for injection. Six mixed standard solutions were prepared: Mixed Standard Solution-1, Mixed Standard Solution-2, Mixed Standard Solution-3, Mixed Standard Solution-4, Mixed Standard Solution-5, and Mixed Standard Solution-6. The concentrations of glycyrrhizin in these six mixed standard solutions were: 0.15, 0.3, 1.5, 7.5, 15, and 30 ng / mL, respectively; the concentrations of isoglycyrrhizin were: 0.05, 0.1, 0.5, 2.5, 5, and 10 ng / mL, respectively; the concentrations of luteolin were: 0.1, 0.2, 1, 5, 10, and 20 ng / mL, respectively; and the concentrations of psoralen were: 0.5, 1, 5, and 6, respectively. 25, 50, and 100 ng / mL. The mixed standard solutions were sequentially injected and detected using the mass spectrometry and liquid chromatography methods described above. A quantitative method was established using the Analytics module in the OS software, and a standard curve was constructed with concentration on the x-axis and peak area on the y-axis. The quantitative ion pairs, retention times, linear ranges, and linear regression equations for the four active ingredients are shown in Table 4 below. The correlation coefficients (r²) for all four components are greater than 0.99, indicating that the four analytes have good linear relationships within their respective ranges and can be used for accurate quantification.

[0035] Table 4. Standard curve information for four types of test objects 2.3 Method Accuracy and Repeatability Analysis Six samples were pretreated as follows: Accurately weigh 0.02 g of the Gubenzhike Granules sample, add 10 μL of a 20 μg / mL mixed standard solution (containing 200 ng / sample for the four analytes), i.e., a sample spiked amount of 10 μg / g. Let stand at room temperature for 10 min, then add 10 mL of 70% methanol-water mixture, vortex to mix, and sonicate at room temperature for 30 min (300w, 40 kHz). Use 70% methanol-water to replenish the lost extraction reagent weight. Centrifuge at 4200 rpm for 10 min at room temperature, carefully collect the supernatant, filter the supernatant through a 0.22 μm organic nylon membrane, and dilute 4-fold (at this point, the theoretical spiked concentration in the sample spiked solution is 5 ng / mL) for analysis. The recovery results are shown in Table 4. The recoveries of glycyrrhizin, isoglycyrrhizin, luteolin, and psoralen were all between 80% and 120%, indicating good method accuracy and the absence of matrix interference, making it suitable for accurate quantification. Meanwhile, the RSD (CV) detected by the four active ingredient mass spectrometry methods was less than 15%, indicating that the sample determination method has good repeatability.

[0036] Table 5. Recovery results of spiked samples of four compounds (n=6) Example 3 Detection and content calculation of active ingredients in Gubenzhike granules Six parallel experiments were conducted. 0.02 g of the Gubenzhike Granules sample was accurately weighed and added to 10 mL of 70% methanol-water mixture. The mixture was vortexed and ultrasonically extracted at room temperature for 30 min (300 W, 40 kHz). The weight of the extraction reagent was replenished with 70% methanol-water. The mixture was centrifuged at 4200 rpm for 10 min at room temperature. The supernatant was carefully collected, filtered through a 0.22 μm organic nylon filter membrane, diluted four times (with 70% methanol-water), and then analyzed.

[0037] The peaks of each ion pair in the sample are shown as follows Figure 3 As shown, the sample contains four compounds: glycyrrhizin, isoglycyrrhizin, luteolin, and psoralen. The ion-pair elution times of glycyrrhizin, isoglycyrrhizin, luteolin, and psoralen in the sample are 2.13, 2.57, 2.10, and 4.52 min, respectively, consistent with the elution times of the four compounds in the standard. Therefore, quantification can be performed using the ion-pair peak areas of the four compounds.

[0038] The contents of the four active ingredients in the Gubenkechuan Granules samples are shown in Table 6. The average contents of glycyrrhizin in the six samples were 45.64 μg / g, isoglycyrrhizin was 6.73 μg / g, luteolin was 0.25 μg / g, and psoralen was 98.55 μg / g.

[0039] Table 6. Content of four active ingredients in Gubenzhike granules samples (n=6) in conclusion: In summary, this invention discloses a UPLC-MS / MS method for simultaneous detection of multiple components in traditional Chinese medicine compound preparations, achieving high-sensitivity detection (0.1 ng / mL) of glycyrrhizin, isoglycyrrhizin, luteolin, and psoralen, while demonstrating good accuracy and repeatability in methodological evaluation. This method has been successfully applied to the first quantitative analysis of key antitussive active ingredients in Gubenzhike granules, obtaining accurate content data for glycyrrhizin, isoglycyrrhizin, luteolin, and psoralen, providing crucial technical support for elucidating the material basis of their antitussive effects and promoting rational clinical use.

[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A qualitative and quantitative method for simultaneous detection of four active ingredients in a Gubenzhike granule sample by UPLC-MS / MS, characterized in that, The four active ingredients are glycyrrhizin, isoglycyrrhizin, luteolin, and psoralen; the method includes the following steps: X1. Pre-treat the sample of Gubenzhike Granules to be tested to obtain the target sample solution; X2. The target sample solution was detected by ultra-high performance liquid chromatography in tandem triple quadrupole mass spectrometry, combined with multiple reaction monitoring (MRM) mode, to achieve qualitative identification and content determination of four active ingredients; The sample pretreatment method was 70% methanol ultrasonic extraction, the liquid chromatography system used was a SCIEX ExionLC AE high-performance liquid chromatography system, and the mass spectrometer was an AB SCIEX 4500.

2. The method according to claim 1, characterized in that, The detection conditions for the ultra-high performance liquid chromatography are as follows: Chromatographic column: A C18 column is selected, preferably the HSS T3 model, with a column length of 100 mm, an inner diameter of 2.1 mm, a particle size of 1.8 μm, and a column temperature of 35℃; Mobile phase: Phase A is 0.1% formic acid solution, and Phase B is acetonitrile; Gradient elution conditions: At 0.00 min, phase A accounts for 70% and phase B accounts for 30%, with a flow rate of 0.3 mL / min; at 3.00 min, the phase A ratio is adjusted to 5% and the phase B ratio is adjusted to 95%, with the flow rate still at 0.3 mL / min; at 5.00 min, the ratio of phase A to phase B is maintained at 5% and the flow rate at 95%, with a flow rate of 0.3 mL / min; at 5.01 min, the initial ratio of phase A to phase B is restored to 70% and the flow rate at 30%, with a flow rate maintained at 0.3 mL / min; at 7.00 min, the ratio of phase A to phase B is maintained at 70% and the flow rate at 30%, with a flow rate of 0.3 mL / min. Injection volume: 5 μL; Sample diluent: 70% methanol-water.

3. The method according to claim 1, characterized in that, The specific pretreatment steps for the sample to be tested are as follows: W1. Accurately weigh 0.02g of the Gubenzhike Granules sample and place it in a centrifuge tube; W2. Add 10 mL of 70% methanol-water to the centrifuge tube, vortex to mix, and then perform ultrasonic extraction at room temperature; W3. After ultrasonic extraction, replenish the weight of the extraction reagent lost due to ultrasonic evaporation with 70% methanol-water solution. W4. Centrifuge the above solution at 4200 rpm for 10 min at room temperature and collect the supernatant; W5. Filter the collected supernatant through a 0.22μm organic nylon filter membrane, preferably a 0.22μm organic nylon filter membrane from the Jinteng brand; W6. Dilute the filtered supernatant with 70% methanol-water by 4 times to obtain the target sample solution, ready for instrument detection.

4. The method according to claim 3, characterized in that The conditions for ultrasonic extraction are: ultrasonic power 300W, ultrasonic frequency 40kHz, and ultrasonic time 30min.

5. The method according to claim 1, characterized in that, The detection conditions for the triple quadrupole mass spectrometer are as follows: Ion source: Electrospray ionization source, scanning mode is negative ion scanning; Ion source parameters: ionization voltage is -4500V, ion source temperature is 550℃, atomizing gas pressure is 50psi, auxiliary heating gas pressure is 55psi, curtain gas pressure is 35psi, collision gas / collision activated dissociation intensity is Medium-9. Multiple reaction monitoring (MRM) conditions: The precursor ion of glycyrrhizin was 255.080, with corresponding daughter ions of 119.010 and 134.930, both with a residence time of 50 ms. The collision voltage and declustering voltage for daughter ion 119.010 were -45.0 V and -70.0 V, respectively, while those for daughter ion 134.930 were -22.0 V and -65.0 V. The precursor ion of isoglycyrrhizin was 255.010, with corresponding daughter ions of 119.010 and 134.940, both with a residence time of 50 ms. The collision voltage and declustering voltage for daughter ion 119.010 were -31.0 V and -50 V, respectively, while those for daughter ion 134.940 were -22.0 V and -50 V. Luteolin... The parent ion is 285.08, and the corresponding daughter ions are 132.970 and 150.930, respectively. The residence time for both is 50 ms. The collision voltage and declustering voltage of daughter ion 132.970 are -48V and -65V, respectively, while those for daughter ion 150.930 are -36V and -65V, respectively. The parent ion for psoralen is 255.180, and the corresponding daughter ion is 172.

000. The residence time is 50 ms, and the collision voltage is -25V and the declustering voltage is -60V. Among the above compounds, the daughter ions labeled for quantitative detection are 134.930 for glycyrrhizin, 119.010 for isoglycyrrhizin, 132.970 for luteolin, and 172.000 for psoralen. The remaining daughter ions are used for qualitative detection.

6. The method according to claim 1, characterized in that, The criteria for qualitative detection are as follows: the retention time of each active ingredient in the target sample solution deviates from the retention time of the standard within ±0.1 min, and the corresponding qualitative ion pair response signal intensity meets the detection requirements; among them, the retention time of glycyrrhizin is 2.1 min, the retention time of isoglycyrrhizin is 2.6 min, the retention time of luteolin is 2.1 min, and the retention time of psoralen is 4.5 min.

7. The method according to claim 1, characterized in that, The quantitative detection calculation method is as follows: First, prepare a mixed standard solution of four active ingredients with six different concentration gradients, and detect it under the chromatographic and mass spectrometric conditions described in claims 2 and 5. Plot a standard curve with the standard solution concentration as the abscissa and the peak area of ​​the corresponding quantitative ion pair as the ordinate, and obtain the linear regression equation. The quantitative ion pair peak area of ​​the target sample solution was then substituted into the linear regression equation, and the contents of the four active ingredients in the Gubenzhike granules sample were calculated by combining the sample weight, extraction volume and dilution factor.

8. The method according to claim 7, characterized in that, The concentration gradient of the mixed standard solution is as follows: Glycyrrhizin: 0.15 ng / mL, 0.3 ng / mL, 1.5 ng / mL, 7.5 ng / mL, 15 ng / mL, 30 ng / mL; Isoliquiritigenin: 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL; Luteolin: 0.1 ng / mL, 0.2 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL; Bakuchiol: 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL; Furthermore, the correlation coefficients of the standard curves for the four active ingredients were all greater than 0.99, indicating a good linear relationship.

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