Method for detecting forskolin, Y-27632 and AM2394 in cell culture biomass and culture solution

Abstract

The invention provides a method for detecting forskolin, Y-27632 and AM2394 in cell culture biomass and a culture solution, and relates to the technical field of food detection. The detection method comprises the following steps: detecting target objects in cell culture biomass and a culture solution by using an ultra-high performance liquid chromatography-tandem mass spectrometry, and controlling a mobile phase A to be ultrapure water, formic acid and ammonium acetate in a detection process; the mobile phase B is methanol or acetonitrile; the detection method can accurately detect forskolin, Y-27632 and AM2394 in the cell culture biomass and the culture solution and perform quantitative analysis, and meanwhile, the detection method is simple and rapid to operate, high in sensitivity and good in repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and in particular to a method for detecting forskolin, Y-27632 and AM2394 in cell culture biomass and culture fluid. Background Art

[0002] As a key component of novel protein products, cell-cultured meat faces significant challenges in its industrialization and commercialization. New technologies and processes, lacking a history of safe use, may pose new food safety risks. The presence of unintended exogenous chemicals (such as forskolin, Y-27632, and AM2394) is crucial for safety assessment, but supporting detection technologies are currently lacking. Forskolin, also known as forskolin, is an adenylate cyclase activator that activates adenylate cyclase (AC) and increases cyclic adenosine monophosphate (cAMP) levels in various tissue cells, thereby regulating cell function. Y-27632, a 4-aminopyridine, plays a crucial role in the separation, adhesion, proliferation, differentiation, and apoptosis of various cell types. AM2394 is also a structurally unique glucokinase activator (GKA) involved in regulating cell function.

[0003] Therefore, there is an urgent need to establish an accurate and reliable detection method for the detection of forskolin, Y-27632 and AM2394 in cell culture biomass and culture medium, so as to provide strong technical support for the supervision of cell cultured meat production and food safety assurance. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides a method for detecting forskolin, Y-27632 and AM2394 in cell culture biomass and culture fluid. The detection method can accurately detect forskolin, Y-27632 and AM2394 in cell culture biomass and culture fluid and perform quantitative analysis. At the same time, the detection method is simple to operate, rapid, highly sensitive and reproducible.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for detecting forskolin, Y-27632, and AM2394 in cell culture biomass and culture fluid, the method comprising the following steps: S1. Prepare standard solutions of forskolin, Y-27632, and AM2394, and then dilute each standard solution into standard intermediate solutions of different concentrations; S2. Ultrasonic extraction of the cell culture biomass and culture fluid using an extraction solvent, followed by centrifugal filtration, is repeated twice, and the filtrates are combined to obtain a test sample; S3. Ultra-high performance liquid chromatography-tandem mass spectrometry was used for detection; the chromatographic conditions were as follows: chromatographic column: ACQUITYUPLC HSS T3, 100 mm × 2.1 mm inner diameter, the particle size of the stationary phase was 1.8 μm; the mobile phase A was ultrapure water, formic acid, and ammonium acetate, wherein the volume percentage of formic acid in mobile phase A was 0.01%-0.1%, and the concentration of ammonium acetate in mobile phase A was 1-5 mM; the mobile phase B was methanol; and the elution mode of the mobile phase was gradient elution.

[0006] Preferably, the solvent for preparing the forskolin standard solution in step S1 is methanol, the solvent for preparing the Y-27632 standard solution is ultrapure water, and the solvent for preparing the AM2394 standard solution is dimethyl sulfoxide.

[0007] Preferably, the solvent used for dilution in step S1 is a mixed solution of methanol, acetonitrile and ultrapure water in a mass ratio of 1:2:3.

[0008] Preferably, the extraction solvent in step S2 is glacial acetonitrile.

[0009] Preferably, the ultrasonic extraction time in step S2 is 15 minutes.

[0010] Preferably, the gradient elution process in step S3 is: 0 min, 5% mobile phase B; 0-1 min, 5%-5% mobile phase B; 1-1.1 min, 5%-60% mobile phase B; 1.1-4 min, 60%-65% mobile phase B; 4-5 min, 65%-95% mobile phase B; 5-5.5 min, 95%-95% mobile phase B; 5.5-6 min, 95%-5% mobile phase B; 6-7.5 min, 5%-5% mobile phase B.

[0011] Preferably, in step S3, the column temperature of the chromatographic column is 35° C. and the flow rate is 0.3 mL / min.

[0012] Preferably, the mass spectrometry conditions in step S3 are: ion source type: electrospray ion source (ESI); scanning mode: positive and negative ion scanning; ion spray voltage: 3,000 V; ion source temperature: 300°C; nebulizing gas flow rate: 3.0 L / min; drying gas flow rate: 10.0 L / min; heating gas flow rate: 10.0 L / min; heating module temperature: 400°C; interface temperature: 100-400°C; desolvation tube temperature: 100-300°C; detection mode: multiple reaction monitoring (MRM).

[0013] The present invention provides a method for detecting forskolin, Y-27632 and AM2394 in cell culture biomass and culture fluid, which has the following advantages over the prior art: The present invention uses an ACQUITY UPLC HSS T3 chromatographic column to detect the contents of forskolin, Y-27632, and AM2394 in cell culture biomass and culture fluid by ultra-performance liquid chromatography-tandem mass spectrometry. By controlling relevant parameters, forskolin, Y-27632, and AM2394 in cell culture biomass and culture fluid can be accurately detected and quantitatively analyzed. At the same time, the detection method is simple to operate, rapid, highly sensitive, and has good reproducibility, and is suitable for popularization and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a detection chart of forskolin in Example 1 of the present invention; Figure 2 This is a detection diagram of Y-27632 in Example 1 of the present invention; Figure 3 This is a detection diagram of AM2394 in Example 1 of the present invention; Figure 4 Graphs showing the test results of different chromatographic columns in the embodiments of the present invention; Figure 5 Graph showing the detection results of different mobile phase systems in the embodiments of the present invention; Figure 6 Graph showing the test results of different acid and salt ratios of mobile phase A in an embodiment of the present invention; Figure 7 Graph showing the detection results of different interface temperatures in an embodiment of the present invention; Figure 8 Graph showing the detection results of the desolvation tube temperature in an embodiment of the present invention. DETAILED DESCRIPTION

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0016] The following specifically describes the pretreatment method, detection method, and application of forskolin, Y-27632, and AM2394 in a cell culture biomass and culture medium according to an embodiment of the present application: The samples, which consist of two matrices, cell culture biomass and culture fluid, were first pretreated. Specifically, the cell culture biomass and culture fluid samples were frozen at -80°C and thawed at 4°C before use. After no solid ice was evident in the samples, they were removed and placed on ice for weighing.

[0017] It should be noted that cell culture biomass and culture medium are rich in nutrients and are very easy to deteriorate once thawed. After thawing, they need to be placed on ice and processed within 3 to 5 minutes.

[0018] (1) Pretreatment methods of cell culture biomass and culture fluid samples: ① Biomass Sample: Vortex the biomass sample and weigh approximately 0.2 g ± 0.02 g (accurate to 0.001 g) into a 2 mL plastic centrifuge tube. Add 1 mL of glacial acetonitrile and homogenize. Extract in an ice-water bath using ultrasound for 15 minutes. Centrifuge at 15,000 rpm at 4°C for 5 minutes. Transfer the entire supernatant to another 2 mL centrifuge tube. Add 1 mL of glacial acetonitrile and homogenize again. Repeat this process. Combine the two extracts and filter through a 0.22 μm organic phase filter for analysis.

[0019] ② Cell culture fluid sample: Vortex the cell culture fluid to mix thoroughly. Pipette 200 μL (accurate to 0.01 μL) of the cell culture fluid into a 2 mL plastic centrifuge tube. Add 1 mL of glacial acetonitrile and homogenize. Extract in an ice-water bath using ultrasound for 15 minutes. Centrifuge at 15,000 rpm at 4°C for 5 minutes. Transfer the entire supernatant to another 2 mL centrifuge tube. Add 1 mL of glacial acetonitrile and homogenize again. Repeat this process. Combine the two extracts and filter through a 0.22 μm organic phase filter for analysis.

[0020] (2) Preparation of standard solution: Prepare 1 mg / L standard solutions of forskolin, Y-27632, and AM2394, respectively. Accurately weigh 10 mg of each standard of forskolin, Y-27632, and AM2394 into a 10 mL volumetric flask. Dissolve the standard in methanol or ultrapure water according to its solubility and dilute to 10 mL. Shake well to prepare 1000 mg / L stock solutions, which are then diluted stepwise to 1 mg / L stock solutions. Store in a -20°C refrigerator.

[0021] The solvent for dissolving the forskolin solid standard is methanol, the solvent for dissolving the Y-27632 solid standard is ultrapure water, and the solvent for dissolving the AM2394 solid standard is dimethyl sulfoxide.

[0022] Accurately pipette 1 mL of each standard stock solution into a 100 mL volumetric flask, dilute to the mark with a solution of methanol:acetonitrile:ultrapure water (1:2:3), shake well, and prepare a standard intermediate working solution with a concentration of 10 μg / mL. Store in a -20°C refrigerator for later use.

[0023] A mixed standard intermediate solution containing three substances was prepared with methanol: acetonitrile: ultrapure water (1:2:3) to prepare a mixed standard working solution with a mass concentration of 0.1-5000µg / L. Each standard solution was prepared before use.

[0024] (3) Instrument parameter setting Triple quadrupole liquid chromatography-mass spectrometry Liquid chromatography conditions: Column: ACQUITY UPLC HSS T3 (100 mm × 2.1 mm (inner diameter), 1.8 μm); Column temperature: 35°C; Flow rate: 0.3 mL / min. Mobile phases included mobile phase A and mobile phase B. Mobile phase A consisted of ultrapure water, formic acid, and ammonium acetate, with the volume percentage of formic acid in mobile phase A being 0.01-0.1% and the concentration of ammonium acetate in mobile phase A being 1-5 mM. Mobile phase B consisted of methanol or acetonitrile. The mobile phase elution method was gradient elution. The specific elution process is as follows: 0 min, 5% mobile phase B; 0-1 min, 5%-5% mobile phase B; 1-1.1 min, 5%-60% mobile phase B; 1.1-4 min, 60%-65% mobile phase B; 4-5 min, 65%-95% mobile phase B; 5-5.5 min, 95%-95% mobile phase B; 5.5-6 min, 95%-5% mobile phase B; 6-7.5 min, 5%-5% mobile phase B.

[0025] The mass spectrometry conditions in ultra-performance liquid chromatography-tandem mass spectrometry include: ion source type: electrospray ion source (ESI); scanning mode: positive and negative ion scanning; ion spray voltage: 3,000 V; ion source temperature: 300°C; nebulizing gas flow rate: 3.0 L / min; drying gas flow rate: 10.0 L / min; heating gas flow rate: 10.0 L / min; heating module temperature: 400°C; interface temperature: 100-400°C; desolvation tube temperature: 100-300°C; detection method: multiple reaction monitoring (MRM).

[0026] Example 1: Detection methods for forskolin, Y-27632, and AM2394 in cell culture biomass or cell culture fluid: Accurately weigh 0.2 g of biomass (accurate to 0.001 g) or 200 μL of culture medium into a 2 mL centrifuge tube. Add 1 mL of glacial acetonitrile (extraction solvent) and sonicate in an ice-water bath for 15 min. Centrifuge the extracted mixed solution at 4°C and 15,000 rpm for 5 min. Repeat the above steps, combine the two supernatants, filter through a 0.22 μm filter membrane, and place in a brown injection vial. Dissolve the solid standard of forskolin, Y-27632, and AM2394 in methanol, ultrapure water, and dimethyl sulfoxide, respectively. Store the prepared standard stock solutions in a -20°C refrigerator. Prepare a precisely 10 μg / mL mixed standard intermediate working solution using a 1:2 methanol:acetonitrile solution. Store the mixed solutions at -20°C until needed. Prepare the mixed solutions of the three substances in a 1:2:3 methanol:acetonitrile:ultrapure water solution to prepare mixed standard intermediate solutions at concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, and 5000 μg / L. Prepare each standard solution immediately before use.

[0027] UHPLC-MS / MS was used for analysis. The mass spectrometry conditions were as follows: ion source: electrospray ionization (ESI); scan mode: positive and negative ion scan; ion spray voltage: 3000 V; ion source temperature: 300°C; nebulizing gas flow rate: 3.0 L / min; drying gas flow rate: 10.0 L / min; heating gas flow rate: 10.0 L / min; heating block temperature: 400°C; interface temperature: 250°C; desolvation tube temperature: 250°C; and detection method: multiple reaction monitoring (MRM). The ion transitions, corresponding collision energies (CE), and plasma characteristics of forskolin, Y-27632, and AM2394 are shown in Table 1.

[0028] Table 1

[0029] Chromatographic column: ACQUITY UPLC HSS T3 column (Waters, USA), column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm; column oven temperature: 35°C; mobile phase: (Phase A) aqueous solution containing 0.01% formic acid and 5 mM ammonium acetate (i.e., the volume fraction of formic acid in mobile phase A is 0.01%, and the concentration of ammonium acetate in mobile phase A is 5 mM); (Phase B) acetonitrile; Method flow rate: 0 min, 5% mobile phase B; 0-1 min, 5%-5% mobile phase B; 1-1.1 min, 5%-60% mobile phase B; 1.1-4 min, 60%-65% mobile phase B; 4-5 min, 65%-95% mobile phase B; 5-5.5 min, 95%-95% mobile phase B; 5.5-6 min, 95%-5% mobile phase B; 6-7.5 min, 5%-5% mobile phase B; sample injection volume: 2 μL.

[0030] For specific test results, see Figure 1 、 Figure 2 and Figure 3 .

[0031] Example 2: The detection method of Example 1 was used with the exception that the ACQUITY UPLC HSS T3 (2.1 mm (inner diameter) × 100 mm, 1.8 µm) column was replaced with an ACQUITY UPLC BEH C18 (2.1 mm (inner diameter) × 100 mm, 1.7 µm) column. The remaining operations and conditions were the same as those of Example 1. The results of the different columns are shown in Table 1. Figure 4 As shown; in Figure 4 (a) represents an ACQUITY UPLC HSS T3 column. Figure 4 (b) represents the ACQUITY UPLC BEHC18 column; Figure 4 1 represents Y-27632, 2 represents AM2394, and 3 represents Forskolin. Figure 4 It can be seen that the ACQUITY UPLC HSS T3 column has strong retention for the compounds, the peaks of the compounds eluting early are sharper, and the compounds are well separated. Therefore, the ACQUITY UPLC HSS T3 (2.1 mm (inner diameter) × 100 mm, 1.8 µm) column was selected for subsequent experiments.

[0032] Example 3: The detection method of Example 1 was used with reference to the above, except that the mobile phase A was ultrapure water and the methanol in the mobile phase B was replaced with acetonitrile. Figure 5 ,in, Figure 5 (a) represents a water / methanol mobile phase system, and (b) represents a water / acetonitrile mobile phase system; according to Figure 5 It can be seen that using methanol as mobile phase B significantly improves the chromatographic peak shape of Y-27632, reducing the half-peak width, making the peak sharper and more sensitive, and also improving the response of forskolin. Therefore, water and methanol were ultimately selected as the preliminary mobile phase system for this experiment.

[0033] Example 4: Referring to the detection method of Example 1 above, the only difference is that the mobile phase system is optimized according to the ratio in Table 2 below.

[0034] Table 2

[0035] Specific results can be found in Figure 6When the mobile phase ratio was a, the relative peak area of ​​forskolin increased significantly, and Y-27632 exhibited a better peak shape after the addition of formic acid. Adding three different salt contents to the mobile phase ratio a revealed that when the mobile phase was f, the relative peak areas of each substance were better, and all targets achieved satisfactory sensitivity. Based on a comprehensive consideration of peak shape and response intensity, 0.01% formic acid-5 mM ammonium acetate in water (phase A) and methanol (phase B) were selected as the mobile phase systems.

[0036] Example 5: The detection method of Example 1 was used with the exception that the interface temperature was optimized. The responses of three substances were measured at interface temperatures of 100°C, 150°C, 200°C, 250°C, 300°C, 350°C, and 400°C. The peak area of ​​each substance at 100°C was set to 100 to examine the effect of different temperatures on the peak area. Specific results can be found in Figure 7 When the interface temperature is at 250°C, the peak area of ​​forskolin increases, and its peak area reaches 1.3 times that at 100°C; and when the interface temperature continues to rise, the peak area of ​​forskolin shows a downward trend. The relative peak area of ​​AM2394 gradually increases with the increase of interface temperature, and there is a significant positive correlation between the two. After the interface temperature exceeds 150°C, the relative peak area of ​​Y-27632 decreases as the interface temperature continues to increase. Among the three substances studied, the absolute value of the response of forskolin is relatively low, at 10 4 Taking into account the responses of the three substances, 250°C was selected as the interface temperature. At this temperature, the peak area of ​​forskolin increased by 1.3 times. Although the relative peak area of ​​AM2394 gradually increased with increasing temperature, the absolute value of the response of forskolin was relatively low. At 250°C, the relative peak areas of the substances did not show a significant decrease, indicating that the detection sensitivity met the detection requirements.

[0037] Example 6: The detection method of Example 1 was used with the exception that the temperature of the desolvation tube was different. The responses of three substances were measured at desolvation tube temperatures of 100°C, 150°C, 200°C, 250°C, and 300°C. The peak area of ​​each substance at 100°C was set to 100 to examine the effect of different temperatures on the peak area. Specific results can be found in Figure 8As the desolvation tube temperature increased, the relative peak areas of Y-27632 and AM2394 decreased, showing a significant negative correlation with temperature. Forskolin, on the other hand, reached its maximum relative peak area at 250°C. Taking comprehensive considerations to ensure a good response for all substances, a desolvation tube temperature of 250°C was ultimately selected. At this temperature, the relative peak areas of the substances did not decrease significantly, and the detection sensitivity met the required detection requirements.

[0038] Example 7: Referring to the detection method of Example 1 above, the only difference is that the extraction solvent is changed to glacial methanol, and the recovery rates of forskolin, Y-27632 and AM2394 in different extraction solvents are detected. The specific results are shown in Table 3: Table 3

[0039] As shown in Table 3, glacial acetonitrile, as the extraction solvent, achieved good recoveries for all three substances, ranging from 82.0% to 110.3%. It also performed best in eliminating solvent effects, improving peak shape, and increasing recoveries, significantly outperforming glacial methanol. Therefore, glacial acetonitrile was selected as the extraction solvent in this study to ensure analytical accuracy and reproducibility.

[0040] Example 8: Methodological evaluation The method used in Example 1 was evaluated based on linearity, limit of detection (LOD), limit of quantification (LOQ), intra-day precision, inter-day precision, recovery, and matrix effects. Accurate quantitative determination was achieved using an external standard method. The LOD and LOQ of forskolin, Y-27632, and AM2394 were determined in cell culture biomass and culture fluid at signal-to-noise ratios (S / N) of 3 and 10, respectively. The precision of the established method was assessed based on repeatability (intra-day precision) and reproducibility (inter-day precision), with the results expressed as relative standard deviations (RSDs). Recovery was used to assess the accuracy of the method. Three recovery spike levels (L (low), M (medium), and H (high)) were designed for recovery. For Y-27632 and AM2394, spike levels of 50 µg / kg, 200 µg / kg, and 500 µg / kg were used for recovery. Because forskolin is present at high levels in cell culture biomass and culture fluid, spike levels of 5 mg / kg, 10 mg / kg, and 50 mg / kg were used for recovery. Matrix effects were evaluated using the slope method, calculated as follows: Slope = (standard curve slope matrix / standard curve slope solvent - 1) × 100 (Formula 1) The linear equation, linear range, and correlation coefficient (r 2), LOD and LOQ are shown in Table 4.

[0041] Table 4

[0042] As shown in Table 4, the linearity of the detection method is good, and the linear correlation coefficient r 2 >0.9985; LODs for forskolin, Y-27632, and AM2394 ranged from 0.1 to 0.6 μg / L, and LOQs from 0.2 to 1.5 μg / L, meeting detection requirements. This method, employing MRM scanning mode, offers high sensitivity and accurate quantitative results, providing technical support for the determination of forskolin, Y-27632, and AM2394 in cell culture biomass and culture fluids.

[0043] The recovery, matrix effect, and precision results of forskolin, Y-27632, and AM2394 in cell culture biomass and culture medium are shown in Table 5 (Recovery and matrix effect of forskolin, Y-27632, and AM2394 in cell culture medium) and Table 6 (Recovery and matrix effect of forskolin, Y-27632, and AM2394 in biomass).

[0044] Table 5

[0045] Table 6

[0046] As shown in Tables 5-6, low-level recoveries ranged from 81.1% to 110.8%, intermediate-level recoveries from 88.4% to 111.5%, and high-level spike recoveries from 85.7% to 116.1%. The intra-day and inter-day reproducibility RSDs were <10%, indicating that this instrumental method and pretreatment method met the methodological evaluation requirements and can be used for the detection of forskolin, Y-27632, and AM2394 in cell culture biomass and culture broth. The matrix effects for forskolin, Y-27632, and AM2394 in cell culture biomass and culture broth ranged from 0.8 to 1.2, indicating that this method exhibited minimal matrix effects and met the requirements for qualitative and quantitative external standardization using solvent-based standards. Therefore, this method is suitable for the qualitative and quantitative detection of forskolin, Y-27632, and AM2394 in cell culture biomass and culture broth.

[0047] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for detecting forskolin, Y-27632 and AM2394 in cell culture biomass and culture medium, characterized in that: The detection method comprises the following steps: S1. Prepare standard solutions of forskolin, Y-27632, and AM2394, and then dilute each standard solution into standard intermediate solutions of different concentrations; S2. Ultrasonic extraction of the cell culture biomass and culture fluid using an extraction solvent, followed by centrifugal filtration, is repeated twice, and the filtrates are combined to obtain a test sample; S3. Detection was performed using ultra-high performance liquid chromatography-tandem mass spectrometry; and the chromatographic conditions were: Chromatographic column: ACQUITY UPLC HSS T3, 100 mm × 2.1 mm inner diameter, stationary phase particle size 1.8 μm; Mobile phase A is ultrapure water, formic acid, and ammonium acetate, wherein the volume percentage of formic acid in mobile phase A is 0.01%-0.1%, and the concentration of ammonium acetate in mobile phase A is 1-5 mM; Mobile phase B was methanol; The elution mode of the mobile phase is gradient elution.

2. The detection method according to claim 1, wherein: In step S1, the solvent for preparing the forskolin standard solution is methanol, the solvent for preparing the Y-27632 standard solution is ultrapure water, and the solvent for preparing the AM2394 standard solution is dimethyl sulfoxide.

3. The detection method according to claim 1, wherein: The solvent used for dilution in step S1 is a mixed solution of methanol, acetonitrile and ultrapure water in a mass ratio of 1:2:

3.

4. The detection method according to claim 1, wherein: The extraction solvent in step S2 is glacial acetonitrile.

5. The detection method according to claim 1, wherein: The time for ultrasonic extraction in step S2 is 15 minutes.

6. The detection method according to claim 1, wherein: The gradient elution process in step S3 is: 0 min, 5% mobile phase B; 0-1 min, 5%-5% mobile phase B; 1-1.1 min, 5%-60% mobile phase B; 1.1-4 min, 60%-65% mobile phase B; 4-5 min, 65%-95% mobile phase B; 5-5.5 min, 95%-95% mobile phase B; 5.5-6 min, 95%-5% mobile phase B; 6-7.5 min, 5%-5% mobile phase B.

7. The detection method according to claim 1, wherein: In step S3, the column temperature of the chromatographic column is 35° C. and the flow rate is 0.3 mL / min.

8. The detection method according to claim 1, wherein: The mass spectrometry conditions in step S3 are: Ion source type: electrospray ion source; Scan mode: positive and negative ion scanning; Ion spray voltage: 3000V; Ion source temperature: 300℃; Nebulizing gas flow rate: 3.0 L / min; Drying gas flow rate: 10.0 L / min; Heating gas flow rate: 10.0 L / min; Heating block temperature: 400°C; Interface temperature: 100-400°C; Desolvation tube temperature: 100-300°C; Detection method: Multiple reaction monitoring.

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