A method for detecting foscorlin, Y-27632 and AM2394 in cell culture biomass and culture medium.

Abstract

This invention provides a method for detecting forscolin, Y-27632, and AM2394 in cell culture biomass and culture medium, relating to the field of food testing technology. The method utilizes ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) to detect the target analytes in cell culture biomass and culture medium. During the detection process, mobile phase A is controlled to be ultrapure water, formic acid, and ammonium acetate; mobile phase B is methanol or acetonitrile. This method can accurately detect and quantitatively analyze forscolin, Y-27632, and AM2394 in cell culture biomass and culture medium. Furthermore, the method is simple, rapid, highly sensitive, and reproducible.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, specifically to a method for detecting foscorlin, Y-27632 and AM2394 in cell culture biomass and culture medium. Background Technology

[0002] As a crucial component of novel proteins, the safety evaluation of cultured meat presents a significant challenge for its industrialization and marketization. The lack of a safe usage history for new technologies and processes may introduce new food safety risks. Among these risks, the addition of unintended chemical substances (such as forsocolin, Y-27632, and AM2394) is key to safety assessment, but currently, supporting detection technologies are lacking. Forsocolin, also known as fusocolin, is an adenylate cyclase activator that increases the concentration of cyclic adenosine monophosphate (cAMP) in various tissue cells by activating adenylate cyclase (AC), thereby regulating cell function. Y-27632 belongs to the 4-aminopyridine class and plays an important role in the separation, adhesion, proliferation, differentiation, and apoptosis of various cell types. AM2394 is also a glucokinase activator (GKA) with a unique structure involved in cell function regulation.

[0003] Therefore, there is an urgent need to establish an accurate and reliable detection method for the detection of foscorlin, 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] To address the shortcomings of existing technologies, this invention provides a method for detecting forscolin, Y-27632, and AM2394 in cell culture biomass and culture medium. This method can accurately detect and quantitatively analyze forscolin, Y-27632, and AM2394 in cell culture biomass and culture medium. Furthermore, this method is simple, rapid, highly sensitive, and has good repeatability.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0006] A method for detecting forscorin, Y-27632, and AM2394 in cell culture biomass and culture medium, the method comprising the following steps:

[0007] S1. Prepare standard solutions of foscorine, Y-27632 and AM2394, and then dilute each standard solution to prepare intermediate standard solutions of different concentrations;

[0008] S2. The cell culture biomass and culture medium were extracted separately by ultrasonic extraction with extraction solvent, followed by centrifugation and filtration. The operation was repeated twice, and the filtrates were combined to obtain the test sample.

[0009] S3. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) was used for detection. The chromatographic conditions were as follows: column: ACQUITYUPLC HSS T3, 100 mm × 2.1 mm inner diameter, stationary phase particle size of 1.8 μm; mobile phase A consisted of ultrapure water, formic acid, and ammonium acetate, with formic acid comprising 0.01%-0.1% by volume and ammonium acetate comprising 1-5 mM by volume; mobile phase B consisted of methanol; and gradient elution was used.

[0010] Preferably, in step S1, the solvent for preparing the fuscalilin 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.

[0011] Preferably, the dilution in step S1 is a mixed solution of methanol, acetonitrile, and ultrapure water in a mass ratio of 1:2:3.

[0012] Preferably, the solvent extracted in step S2 is glacial acetonitrile.

[0013] Preferably, the ultrasonic extraction time in step S2 is 15 minutes.

[0014] Preferably, the gradient elution process in step S3 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.

[0015] Preferably, in step S3, the column temperature of the chromatographic column is 35°C and the flow rate is 0.3 mL / min.

[0016] Preferably, the mass spectrometry conditions in step S3 are as follows: ion source type: electrospray ionization (ESI); scanning mode: positive and negative ion scanning; ion spray voltage: 3000 V; ion source temperature: 300℃; nebulizer 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℃; interface temperature: 100-400℃; desolvation tube temperature: 100-300℃; detection mode: multiple reaction monitoring (MRM).

[0017] This invention provides a method for detecting forscorin, Y-27632, and AM2394 in cell culture biomass and culture medium, which has the following advantages compared with the prior art:

[0018] This invention utilizes an ACQUITY UPLC HSS T3 column and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) to detect the contents of forscorin, Y-27632, and AM2394 in cell culture biomass and culture medium. By controlling relevant parameters, the method can accurately detect and quantitatively analyze forscorin, Y-27632, and AM2394 in cell culture biomass and culture medium. Furthermore, this detection method is simple, rapid, highly sensitive, and reproducible, making it suitable for widespread application. Attached Figure Description

[0019] Figure 1 This is a detection chart of forscolin in Embodiment 1 of the present invention;

[0020] Figure 2 This is the detection image of Y-27632 in Embodiment 1 of the present invention;

[0021] Figure 3 This is a detection image of AM2394 in Embodiment 1 of the present invention;

[0022] Figure 4 The images show the detection results of different chromatographic columns in the embodiments of the present invention;

[0023] Figure 5 The graph shows the detection results of different mobile phase systems in the embodiments of the present invention;

[0024] Figure 6 The graph shows the detection results of different acid and salt ratios in mobile phase A in this embodiment of the invention.

[0025] Figure 7 The graph shows the detection results of different interface temperatures in an embodiment of the present invention.

[0026] Figure 8 This is a graph showing the detection results of the desolvation tube temperature in an embodiment of the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] The following describes in detail a pretreatment method, detection method, and application of forsocolin, Y-27632, and AM2394 in cell culture biomass and culture medium according to embodiments of this application:

[0029] The sample contains two substrates: cell culture biomass and culture medium. Pretreatment is performed first. Specifically, the cell culture biomass and culture medium samples are frozen at -80℃ and thawed at 4℃ before use until no obvious solid ice particles remain. Then, the samples are removed and placed on ice for weighing.

[0030] It should be noted that cell culture biomass and culture medium are rich in nutrients and are very prone to spoilage once thawed. After thawing, they should be placed on ice and processed within 3-5 minutes.

[0031] (1) Pretreatment methods for cell culture biomass and culture medium samples:

[0032] ① Biomass Sample: Vortex the biomass to mix thoroughly. Weigh approximately 0.2 g ± 0.02 g (accurate to 0.001 g) of biomass sample into a 2 mL plastic centrifuge tube. Add 1 mL of ice-cold acetonitrile, homogenize, and extract by ultrasonication in an ice-water bath for 15 min. Centrifuge at 15000 r / min for 5 min at 4℃. Transfer all supernatant to another 2 mL centrifuge tube. Add another 1 mL of ice-cold acetonitrile to the sample, homogenize, and repeat the above operation. Combine the two extracts and filter through a 0.22 μm organic phase filter membrane for analysis.

[0033] ② Cell culture medium sample: Vortex the cell culture medium to mix well, pipette 200 μL (accurate to 0.01 µL) of the cell culture medium sample into a 2 mL plastic centrifuge tube, add 1 mL of ice-cold acetonitrile, homogenize, and sonicate in an ice-water bath for 15 min. Centrifuge at 15000 r / min for 5 min at 4℃, and transfer all supernatant to another 2 mL centrifuge tube. Add another 1 mL of ice-cold acetonitrile to the sample, homogenize, and repeat the above operation. Combine the two extracts, filter through a 0.22 μm organic phase filter membrane, and analyze using the centrifuge.

[0034] (2) Preparation of standard solutions:

[0035] Prepare 1 mg / L standard solutions of forscorin, Y-27632 and AM2394 respectively. Accurately weigh 10 mg of forscorin, Y-27632 and AM2394 standards into 10 mL volumetric flasks. Dissolve the standards in solvents such as methanol or ultrapure water according to their solubility and dilute to 10 mL. Shake well and prepare 1000 mg / L stock solutions respectively. Then dilute stepwise to 1 mg / L stock solutions and store in a -20℃ refrigerator.

[0036] The solvent for the fuscaline solid standard is methanol, the solvent for the Y-27632 solid standard is ultrapure water, and the solvent for the AM2394 solid standard is dimethyl sulfoxide.

[0037] Accurately pipette 1 mL of each standard stock solution into a 100 mL volumetric flask, dilute to the mark with a methanol:acetonitrile:ulpure water (1:2:3) solution, shake well, and prepare a standard intermediate working solution with a concentration of 10 μg / mL. Store at -20℃ for later use.

[0038] The mixed standard intermediate solution containing the three substances was prepared into mixed standard working solutions with a mass concentration of 0.1-5000 µg / L using a methanol:acetonitrile:ultrapure water (1:2:3) solution. Each standard solution was prepared fresh for each use.

[0039] (3) Instrument parameter settings

[0040] Triple quadrupole liquid chromatography-mass spectrometry

[0041] Liquid chromatography conditions: Column: ACQUITY UPLC HSS T3 (100 mm × 2.1 mm (inner diameter), 1.8 μm); Column temperature: 35℃; Flow rate: 0.3 mL / min. The mobile phases consisted of mobile phase A and mobile phase B. Mobile phase A comprised ultrapure water, formic acid, and ammonium acetate, with formic acid comprising 0.01-0.1% by volume and ammonium acetate comprising 1-5 mM. Mobile phase B consisted of methanol or acetonitrile. The elution method was gradient elution. The specific elution process is as follows:

[0042] 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.

[0043] The mass spectrometry conditions in ultra-high performance liquid chromatography-tandem mass spectrometry include: ion source type: electrospray ionization (ESI); scanning mode: positive and negative ion scanning; ion spray voltage: 3000 V; ion source temperature: 300 ℃; nebulizer 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 ℃; interface temperature: 100-400 ℃; desolvation tube temperature: 100-300 ℃; detection mode: multiple reaction monitoring (MRM).

[0044] Example 1:

[0045] Methods for detecting forsocolin, Y-27632, and AM2394 in cell culture biomass or cell culture medium:

[0046] Accurately weigh 0.2 g of biomass (accurate to 0.001 g) or 200 μL of culture medium into 2 mL centrifuge tubes, accurately add 1 mL of ice-cold acetonitrile (extraction solvent), and sonicate in an ice-water bath for 15 min; centrifuge the extracted mixture at 4 °C and 15000 rpm for 5 min, repeat the above steps, combine the two supernatants, filter through a 0.22 μm filter membrane into a brown vial;

[0047] Foscorine solid standard, Y-27632 solid standard, and AM2394 solid standard were dissolved in methanol, ultrapure water, and dimethyl sulfoxide, respectively. The prepared standard stock solutions were stored at -20°C. A 10 μg / mL mixed standard intermediate working solution was precisely prepared from each of the above standard stock solutions using a methanol:acetonitrile (1:2) solution, and stored at -20°C for later use. Mixed standard intermediate solutions with mass concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, and 5000 μg / L were prepared using a methanol:acetonitrile:ulpure water (1:2:3) solution. Each standard solution was prepared fresh before use.

[0048] UHPLC-MS / MS was used for detection and analysis. The mass spectrometry conditions were as follows: ion source type: electrospray ionization (ESI); scanning mode: positive and negative ion scanning; ion spray voltage: 3000 V; ion source temperature: 300 ℃; nebulizer 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 ℃; interface temperature: 250 ℃; desolvation tube temperature: 250 ℃; detection mode: multiple reaction monitoring (MRM). The ion pairs, corresponding collision energies (CE), and inductively coupled plasma mass spectrometry conditions of fossiline, Y-27632, and AM2394 are shown in Table 1.

[0049] Table 1

[0050]

[0051] Chromatographic column: ACQUITY UPLC HSS T3 column (Waters Corporation, USA), column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm; column oven temperature: 35℃; mobile phase: (phase A) aqueous solution containing 0.01% formic acid and 5 mM ammonium acetate (i.e., formic acid volume fraction in mobile phase A is 0.01%, and ammonium acetate concentration in mobile phase A is 5 mM), (phase B) acetonitrile;

[0052] 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.

[0053] For detailed test results, please see [link / details]. Figure 1 , Figure 2 and Figure 3 .

[0054] Example 2:

[0055] The detection method described in Example 1 above was followed, except 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. All other operations and conditions remained the same as in Example 1, and the results for different columns are shown below. Figure 4 As shown;

[0056] in Figure 4 (a) indicates an ACQUITY UPLC HSS T3 column. Figure 4 (b) indicates an ACQUITY UPLC BEHC18 column; Figure 4 In the diagram, 1 represents Y-27632, 2 represents AM2394, and 3 represents Fuscolin. According to... Figure 4 It can be seen that the ACQUITY UPLC HSS T3 column has strong retention of compounds, and the peaks of compounds that elute early are sharper. The separation of each compound is better. Therefore, the ACQUITY UPLC HSS T3 (2.1 mm (inner diameter) × 100 mm, 1.8 µm) column was selected for subsequent experiments.

[0057] Example 3:

[0058] The detection method described in Example 1 above was used, with the only difference being that mobile phase A was ultrapure water, and methanol in mobile phase B was replaced with acetonitrile. See the results below. Figure 5 ,in, Figure 5 In the diagram, (a) represents the water / methanol mobile phase system and (b) represents the water / acetonitrile mobile phase system.

[0059] according to Figure 5 It is evident that using methanol as mobile phase B significantly improves the peak shape of Y-27632, reducing the half-peak width, making the peak sharper, and increasing sensitivity, while also enhancing the response of foscorine. Therefore, water and methanol were ultimately chosen as the initial mobile phase system for this experiment.

[0060] Example 4:

[0061] The detection method described in Example 1 above is the same, except that the mobile phase system is optimized according to the proportions in Table 2 below.

[0062] Table 2

[0063]

[0064] For details, please see [link / details]. Figure 6 When the mobile phase ratio was 'a', the relative peak area of ​​foscorine increased significantly, and Y-27632 showed a better peak shape after the addition of formic acid. Adding three different amounts of salt 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 target analytes achieved satisfactory sensitivity. Considering both peak shape and response intensity, 0.01% formic acid-5 mM ammonium acetate aqueous solution (phase A) and methanol (phase B) were selected as the mobile phase system.

[0065] Example 5:

[0066] Referring to the detection method in Example 1 above, the only difference is that the interface temperature is optimized. The responses of three substances at interface temperatures of 100℃, 150℃, 200℃, 250℃, 300℃, 350℃, and 400℃ were measured. The peak area of ​​each substance at 100℃ was set to 100 to examine the effect of different temperatures on the peak area.

[0067] For details, please see [link / details]. Figure 7 When the interface temperature was 250℃, the peak area of ​​forscoringin increased, reaching 1.3 times that at 100℃; however, as the interface temperature continued to rise, the peak area of ​​forscoringin showed a decreasing trend. The relative peak area of ​​AM2394 gradually increased with increasing interface temperature, showing a significant positive correlation. The relative peak area of ​​Y-27632 decreased with further increases in interface temperature after exceeding 150℃. Among the three substances studied, forscoringin had a relatively low absolute response value, located at 10...4 Order of magnitude. Considering the response of the three substances, 250℃ was selected as the interface temperature. At this temperature, the peak area of ​​forscorin reached 1.3 times that of forscorin. Although the relative peak area of ​​AM2394 gradually increased with increasing temperature, the absolute value of forscorin's response was relatively low. Moreover, the relative peak areas of each substance did not decrease significantly at 250℃, and the detection sensitivity met the detection requirements.

[0068] Example 6:

[0069] The detection method of Example 1 above was used, with the only difference being the temperature of the desolventizing tube. The responses of three substances were measured at desolventizing 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.

[0070] For details, please see [link / details]. Figure 8 As the temperature of the desolventizing tube increased, the relative peak areas of substances Y-27632 and AM2394 decreased, showing a significant negative correlation with temperature. Forscorin exhibited the largest relative peak area at 250℃. Considering all factors to ensure good response for all substances, a desolventizing tube temperature of 250℃ was ultimately chosen. Under this temperature condition, the relative peak areas of all substances did not decrease significantly, and the detection sensitivity met the corresponding detection requirements.

[0071] Example 7:

[0072] The detection method described in Example 1 above was used, except that the extraction solvent was changed to icy methanol. The recoveries of different extractants, namely foscorine, Y-27632, and AM2394, were detected, and the specific results are shown in Table 3.

[0073] Table 3

[0074]

[0075] 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%. Furthermore, it demonstrated the best performance in eliminating solvent effects, improving peak shape, and increasing recovery rates, significantly outperforming glacial methanol. Therefore, glacial acetonitrile was selected as the extraction solvent in this study to ensure the accuracy and repeatability of the analysis.

[0076] Example 8:

[0077] Methodological evaluation

[0078] The method of Example 1 was evaluated by linearity, limit of detection (LOD), limit of quantitation (LOQ), intra-day precision, inter-day precision, recovery, and matrix effect. Accurate quantification was performed using the external standard method. LOD and LOQ of forscorin, Y-27632, and AM2394 in cell culture biomass and culture medium were determined with 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), and the results are expressed as relative standard deviation (RSD). Recovery was used to evaluate the accuracy of the method. Three spiking levels (L (low), M (medium), and H (high)) were designed for recovery evaluation. Y-27632 and AM2394 were spiked at 50 µg / kg, 200 µg / kg, and 500 µg / kg for recovery. Since the cell culture biomass and culture medium contained high levels of foscorlin, spiking at 5 mg / kg, 10 mg / kg, and 50 mg / kg were used for recovery. The slope method was used to evaluate the matrix effect. The formula for the slope method is:

[0079] Slope = (Standard curve slope matrix / Standard curve slope solvent - 1) × 100 (Equation 1)

[0080] The linear equation, linear range, and correlation coefficient (r) of the method 2 (LOD and LOQ are shown in Table 4.)

[0081] Table 4

[0082]

[0083] As shown in Table 4, the detection method exhibits good linearity, with the linear correlation coefficient r0 being [value missing]. 2 The LOD (Level of Detection) of forscorin, Y-27632, and AM2394 is >0.9985; the LOD of forscorin, Y-27632, and AM2394 is between 0.1 and 0.6 μg / L, and the LOQ (Level of Detection) is between 0.2 and 1.5 μg / L, which meets the detection requirements. This method uses MRM (Medium-Range Resonance) scanning mode, which has high sensitivity and accurate quantitative results, and can provide technical support for the detection of forscorin, Y-27632, and AM2394 in cell culture biomass and culture medium.

[0084] The recovery rates, matrix effects, and precision results of forscorin, Y-27632, and AM2394 in cell culture biomass and culture medium are shown in Table 5 (Recovery rates and matrix effects of forscorin, Y-27632, and AM2394 in cell culture medium) and Table 6 (Recovery rates and matrix effects of forscorin, Y-27632, and AM2394 in biomass).

[0085] Table 5

[0086]

[0087] Table 6

[0088]

[0089] As shown in Tables 5-6, the recoveries at low levels ranged from 81.1% to 110.8%, those at intermediate levels from 88.4% to 111.5%, and those at high levels from 85.7% to 116.1%. The intra-day and inter-day reproducibility RSDs were <10%, indicating that the instrumental method and pretreatment method meet the methodological evaluation criteria and can be used to detect forscolin, Y-27632, and AM2394 in cell culture biomass and culture medium. The matrix effects of forscolin, Y-27632, and AM2394 in cell culture biomass and culture medium ranged from 0.8 to 1.2, indicating that the matrix effect of this method is not significant, meeting the conditions for qualitative and quantitative determination using external standard methods with solvent-based standards. Therefore, this method is suitable for the qualitative and quantitative detection of forscolin, Y-27632, and AM2394 in cell culture biomass and culture medium.

[0090] 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 forscorin, Y-27632, and AM2394 in cell culture biomass and culture medium, characterized in that, The detection method includes the following steps: S1. Prepare standard solutions of foscorine, Y-27632 and AM2394, and then dilute each standard solution to prepare intermediate standard solutions of different concentrations; S2. The cell culture biomass and culture medium were extracted separately by ultrasonic extraction with extraction solvent, followed by centrifugation and filtration. The operation was repeated twice, and the filtrates were combined to obtain the test sample. S3. Detection was performed using ultra-high performance liquid chromatography-tandem mass spectrometry; and the chromatographic conditions were as follows: Column: ACQUITY UPLC HSS T3, 100 mm × 2.1 mm inner diameter, stationary phase particle size 1.8 μm; Mobile phase A consists of 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 is methanol; Furthermore, the elution method of the mobile phase is gradient elution.

2. The detection method according to claim 1, characterized in that: In step S1, the solvent for preparing the fuscalilin 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, characterized in that: The 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, characterized in that: The solvent extracted in step S2 is glacial acetonitrile.

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

6. The detection method according to claim 1, characterized in that: The gradient elution process in step S3 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.

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

8. The detection method according to claim 1, characterized in that: The mass spectrometry conditions in step S3 are as follows: Ion source type: Electrospray ion source; Scanning mode: Positive and negative ion scanning; Ion spray voltage: 3000V; Ion source temperature: 300℃; Atomizing 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℃; Interface temperature: 100-400℃; Desolventizing tube temperature: 100-300℃; Detection method: Multiple reaction monitoring.

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