A qualitative analysis method of ethinyl estradiol and its degradation products

Abstract

The application belongs to the technical field of analytical chemistry, and discloses a qualitative analysis method of ethinyl estradiol and degradation products thereof, which comprises the following steps: determining an accurate mass database and a mass spectrum library of the ethinyl estradiol and degradation products thereof to be detected by using an ultra-high pressure liquid chromatography-quadrupole-electrical field orbitrap high resolution mass spectrometer; detecting the sample by using the ultra-high pressure liquid chromatography-quadrupole-electrical field orbitrap high resolution mass spectrometer; and comparing and analyzing the detection result of the sample with the established accurate mass database and mass spectrum library, so that when the detection result matches the information of the accurate mass database and mass spectrum library, it is determined that the ethinyl estradiol and degradation products thereof to be detected are detected in the sample. The application can realize high-precision qualitative analysis of the sample to be detected by searching for parent ions and daughter ions of the ethinyl estradiol and degradation products thereof by using the ultra-high pressure liquid chromatography-quadrupole-electrical field orbitrap high resolution mass spectrometer, and establishing a high-resolution database.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of analytical chemistry, and particularly relates to a qualitative analysis method of ethinyl estradiol and degradation products thereof. BACKGROUND

[0002] Environmental estrogens are a typical class of environmental endocrine disruptors (endocrine disrupting chemicals, EDCs), which have extremely strong endocrine disrupting effects and biological activities. They are frequently detected in sewage treatment plants, and have attracted widespread attention in recent decades due to their persistence, high degradation difficulty, continuous discharge, and adverse effects on humans and ecosystems. Estrogen (E1), estradiol (E2), and ethinyl estradiol (EE2) can have obvious effects on organisms at very low concentrations (1.0 ng / L), have serious teratogenic and carcinogenic effects, can induce feminization of male individuals, and can cause female tumors, cancer, male infertility, and precocious puberty in children. Microalgae, as eukaryotic organisms that can perform photosynthesis, have obvious degradation effects on ethinyl estradiol. However, there is a lack of research on the qualitative analysis of ethinyl estradiol degradation products in microalgae. The liquid chromatography-mass spectrometry instrument used in the traditional detection of ethinyl estradiol degradation products in microalgae has low resolution and insufficient accuracy, and cannot accurately qualify the degradation products, which are still in the prediction stage. SUMMARY

[0003] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a qualitative analysis method of ethinyl estradiol and degradation products thereof, which can accurately qualify the degradation products of ethinyl estradiol in microalgae.

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a qualitative analysis method of ethinyl estradiol and degradation products thereof, which can accurately qualify the degradation products of ethinyl estradiol in microalgae.

[0005] Specifically, a qualitative analysis method of ethinyl estradiol and degradation products thereof includes the following steps:

[0006] Establishing an accurate mass database and a mass spectrum library of the ethinyl estradiol to be tested and degradation products thereof: configuring standard solutions of the ethinyl estradiol to be tested and degradation products thereof, and determining an accurate mass database and a mass spectrum library of the ethinyl estradiol to be tested and degradation products thereof by using an ultra-high pressure liquid chromatography-quadrupole-electric field orbitrap high-resolution mass spectrometer;

[0007] Sample detection: adding ethinyl estradiol to microalgae, and then culturing in a culture medium, mixing the algal liquid with a solvent, crushing the algal cells, and then separating the algal cells by solid phase extraction to obtain a sample; and detecting the sample by using an ultra-high pressure liquid chromatography-quadrupole-electric field orbitrap high-resolution mass spectrometer;

[0008] Results analysis: The detection results of the sample were compared with the established precise mass database and mass spectrometry library. When the detection results matched the information in the precise mass database and mass spectrometry library, it was determined that the analyte ethinylestradiol and its degradation products were detected in the sample.

[0009] Preferably, in the qualitative analysis method of ethinylestradiol and its degradation products according to the present invention, the ethinylestradiol degradation products include β-estradiol diacetate, 17-acetate estradiol, estriol, estrone, estradiol, 2-hydroxyestradiol, and 16α-hydroxyestradiol, among β-estradiol diacetate, estrone, estradiol, and 16α-hydroxyestradiol.

[0010] In the qualitative analysis method of ethinylestradiol and its degradation products described in this invention, the ethinylestradiol degradation products include diacetylenol, β-estradiol 17-acetate, estriol, estrone, estradiol, 2-hydroxyestradiol and 16α-hydroxyestradiol.

[0011] Preferably, in the qualitative analysis method for ethinylestradiol and its degradation products according to the present invention, the precise mass database includes the chromatographic retention time of each ethinylestradiol and its degradation product to be tested, and the precise mass numbers of a parent ion and a daughter ion with a high response intensity after applying different collision energies to the ethinylestradiol and its degradation product to be tested; the mass spectrometry library includes secondary mass spectra generated after applying different collision energies to the ethinylestradiol and its degradation product to be tested.

[0012] Preferably, in the qualitative analysis method for ethinylestradiol and its degradation products according to the present invention, the chromatographic conditions of the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometer are as follows:

[0013] Column: Shim-pack Scepter C18-120 (2.1×100 mm, 1.9 μm);

[0014] Mobile phase A is: 0.1 wt% formic acid aqueous solution;

[0015] Mobile phase B is: LC-MS grade methanol;

[0016] Column temperature: 35 ℃;

[0017] Flow rate: 0.3 mL / min;

[0018] Injection volume: 2 μL;

[0019] Elution method: gradient elution.

[0020] Preferably, in the qualitative analysis method for ethinylestradiol and its degradation products according to the present invention, the gradient elution procedure is as follows:

[0021] At 0 minutes, mobile phase A was 60% and mobile phase B was 40%.

[0022] At 10.6 minutes, mobile phase A was 35% and mobile phase B was 65%.

[0023] At 16.5 minutes, mobile phase A was 0% and mobile phase B was 100%.

[0024] At 19.25 minutes, mobile phase A was 0%, and mobile phase B was 100%.

[0025] At 19.35 minutes, mobile phase A was 60% and mobile phase B was 40%.

[0026] After 23 minutes, mobile phase A was 60% and mobile phase B was 40%.

[0027] Preferably, in the qualitative analysis method for ethinylestradiol and its degradation products according to the present invention, the mass spectrometry ion source conditions of the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometer are as follows:

[0028] Electrospray ionization source;

[0029] Negative ion mode;

[0030] Spray voltage: 3.5 kV;

[0031] Capillary temperature: 25 ℃;

[0032] Heater temperature: 50 ℃;

[0033] Sheath gas flow rate: 33 arb;

[0034] Auxiliary gas flow rate: 15 arb;

[0035] Scavenging flow rate: 2 arb;

[0036] Ion lens voltage frequency: 55.0.

[0037] Preferably, in the qualitative analysis method for ethinylestradiol and its degradation products according to the present invention, the mass spectrometry detection conditions of the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometer are as follows:

[0038] Scanning mode: Level 1 full scan / Data-dependent level 2 daughter ion scan;

[0039] Scan range: 200-350 m / z;

[0040] Level 1 full scan: resolution of 70,000, maximum orbital sink capacity of 3×10⁶, maximum orbital sink injection time of 100 ms;

[0041] Data-dependent secondary ion scanning: Target ion list is enabled, with parameters consistent with those in the precise mass database. Resolution is 17500, maximum orbital trap capacity is 1×10⁵, maximum orbital trap injection time is 50 ms, number of cycles is 5, isolation window is 4.0 m / z, collision energy mode and energy values ​​are set to NCE: 15 eV, NCE: 25 eV, NCE: 30 eV, minimum orbital trap capacity is 8×10³, and dynamic exclusion time is 10 s.

[0042] Preferably, the specific process of establishing an accurate quality database of ethinylestradiol and its degradation products includes the following steps:

[0043] Standard solutions of ethinylestradiol and its degradation products at a concentration of 1000 μg / L were prepared and directly injected into a high-resolution mass spectrometer (HPLC-quadrupole-electrostatic field orbital trap) with ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap. Analysis was performed in both positive and negative ion modes. The negative ion mode was selected as the scanning mode. The precise mass number of the parent ion of the corresponding analyte was determined, and the precise mass number of the daughter ion with the highest response intensity was selected as the preset daughter ion. The precise mass number of the parent ion and the preset daughter ion were input into the mass tracking module, and the collision energy modes CE and NCE were switched to optimize the best CE and NCE and monitor the preset daughter ion. The final daughter ion was confirmed to be the daughter ion in the precise mass database.

[0044] In the above process, key mass spectrometry parameters such as electrospray voltage, ion source temperature, sheath gas pressure, and resolution were optimized respectively;

[0045] A mixed standard solution of ethinylestradiol and its degradation products with a concentration of 1000 μg / L was prepared. The ultra-high pressure liquid chromatography separation conditions were optimized, and the chromatographic retention time of each compound was obtained by chromatographic separation.

[0046] Establish a precise mass database: Input the name, molecular formula, Chemical Abstracts number, precise mass number of the parent ion, precise mass number of the daughter ion, chromatographic retention time, and optimal collision energy for each compound.

[0047] Preferably, the specific process of establishing a mass spectrometry library of the ethinylestradiol to be tested and its degradation products includes the following steps:

[0048] Standard solutions of ethinylestradiol and its degradation products with a concentration of 1000 μg / L were prepared and analyzed by ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry. The target compounds were broken down to obtain secondary mass spectra of each compound. All secondary mass spectra were input and stored to obtain a mass spectral library of all the compounds to be tested.

[0049] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0050] This invention leverages the superior resolution of Orbitrap electrostatic field technology to easily achieve high-accuracy mass number determination, with a mass number precision of less than 1 ppm for typical analytes. Using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry (UPLC-Q-Exactive Orbitrap MS), precursor and daughter ions of ethinylestradiol and its degradation products are identified, and a high-resolution mass spectrometry database is established. This enables highly accurate qualitative analysis of ethinylestradiol degradation products in microalgae, providing more precise data support for research on ethinylestradiol degradation in microalgae. Attached Figure Description

[0051] Figure 1 These are the chromatograms and mass spectra of ethinylestradiol in a standard solution;

[0052] Figure 2 These are the chromatograms and mass spectra of diacetylenol in a standard solution;

[0053] Figure 3 These are the chromatograms and mass spectra of β-estradiol 17-acetate in a standard solution;

[0054] Figure 4 These are the chromatograms and mass spectra of estriol in a standard solution;

[0055] Figure 5 These are the chromatograms and mass spectra of estrone in a standard solution;

[0056] Figure 6 These are the chromatograms and mass spectra of estradiol in a standard solution;

[0057] Figure 7 These are the chromatograms and mass spectra of 2-hydroxyestrone in a standard solution;

[0058] Figure 8 These are the chromatograms and mass spectra of 16α-hydroxyestrone in a standard solution;

[0059] Figure 9 These are the chromatograms and mass spectra of β-estradiol 17-acetate in the sample;

[0060] Figure 10 These are the chromatograms and mass spectra of estrone in the sample;

[0061] Figure 11 These are the chromatograms and mass spectra of estradiol in the sample;

[0062] Figure 12 This is a detailed implementation method for the chromatogram and mass spectrum of 16α-hydroxyestrone in the sample. Detailed Implementation

[0063] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0064] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0065] The ethinylestradiol and its degradation products (diacetylenol, β-estradiol 17-acetate, estriol, estrone, estradiol, 2-hydroxyestradiol, 16α-hydroxyestradiol) used in the following examples were purchased from Zhongke Quality Inspection Biotechnology Co., Ltd.

[0066] Example 1

[0067] A qualitative analysis method for ethinylestradiol and its degradation products includes the following steps:

[0068] (1) Establishment of a precise mass database and mass spectrometry library

[0069] The precise mass database includes the chromatographic retention times of each analyte ethinylestradiol and its degradation products, as well as the precise mass numbers of a precursor ion and the daughter ions with higher response intensity after applying different collision energies to the analyte ethinylestradiol and its degradation products; the mass spectrometry library includes secondary mass spectra generated after applying different collision energies to the analyte ethinylestradiol and its degradation products.

[0070] (1.1) The process of establishing a precise quality database includes the following steps:

[0071] Standard solutions of ethinylestradiol and its degradation products at a concentration of 1000 μg / L were prepared and directly injected into a high-resolution mass spectrometer (HPLC-quadrupole-electrostatic field orbital trap) with ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap. Analysis was performed in both positive and negative ion modes. The negative ion mode was selected as the scanning mode. The precise mass number of the parent ion of the corresponding analyte was determined, and the precise mass number of the daughter ion with the highest response intensity was selected as the preset daughter ion. The precise mass number of the parent ion and the preset daughter ion were input into the mass tracking module, and the collision energy modes CE and NCE were switched to optimize the best CE and NCE and monitor the preset daughter ion. The final daughter ion was confirmed to be the daughter ion in the precise mass database.

[0072] In the above process, key mass spectrometry parameters such as electrospray voltage, ion source temperature, sheath gas pressure, and resolution were optimized respectively;

[0073] A mixed standard solution of ethinylestradiol, diacetylenol, β-estradiol 17-acetate, estriol, estrone, estradiol, 2-hydroxyestradiol and 16α-hydroxyestradiol with a concentration of 1000 μg / L was prepared, and the chromatographic retention time of each compound was obtained by chromatographic separation.

[0074] The chromatographic conditions were as follows: column: Shim-pack Scepter C18-120 (2.1×100 mm, 1.9 μm); mobile phase A: 0.1 wt% formic acid aqueous solution; mobile phase B: LC-MS grade methanol; column temperature: 35 ℃; flow rate: 0.3 mL / min; injection volume: 2 μL; elution gradient is shown in Table 1.

[0075] Table 1

[0076]

[0077] Enter the name, molecular formula, Chemical Abstracts Service number, exact mass number of the parent ion, exact mass number of the daughter ion, chromatographic retention time, and optimal collision energy for each compound.

[0078] (1.2) The process of establishing a mass spectrometry library specifically includes the following steps:

[0079] Standard solutions of ethinylestradiol, norethindrone diacetate, β-estradiol 17-acetate, estriol, estrone, estradiol, 2-hydroxyestradiol, and 16α-hydroxyestradiol were prepared at a concentration of 1000 μg / L. These solutions were then directly injected into a high-resolution mass spectrometer (HPLC-quadrupole-electrostatic field orbital trap) for analysis. A series of different collision energies were set to break down the target compounds, obtaining secondary mass spectra for each compound. All secondary mass spectra were input and stored to obtain a mass spectral library of all the analytes.

[0080] A mixed standard solution of ethinylestradiol and its degradation products with a concentration of 1000 μg / L was prepared. The ultra-high pressure liquid chromatography (UHPLC) separation conditions were optimized, and the chromatographic retention time of each compound was obtained through chromatographic separation.

[0081] Establish a precise mass database: Input the name, molecular formula, Chemical Abstracts number, precise mass number of the parent ion, precise mass number of the daughter ion, chromatographic retention time, and optimal collision energy for each compound.

[0082] The mass spectrometry ion source conditions were as follows: electrospray ionization source; negative ion mode; spray voltage: 3.5 kV; capillary temperature: 25 ℃; heater temperature: 50 ℃; sheath gas flow rate: 33 arb; auxiliary gas flow rate: 15 arb; scavenging gas flow rate: 2 arb; ion lens voltage frequency: 55.0.

[0083] Mass spectrometry detection conditions: Scanning mode: Level 1 full scan / data-dependent secondary daughter ion scan; Scanning range: 200-350 m / z; Level 1 full scan: resolution 70000, maximum orbital trap capacity 3×10⁻⁶ m / z. 6 The maximum injection time in the orbital trap is 100 ms; data depends on secondary daughter ion scanning: a target ion list is enabled, and the parameters in the list (mass number, molecular formula, scan mode, collision energy) are consistent with the corresponding parameters in the precise mass database, with a resolution of 17500 and a maximum orbital trap capacity of 1×10⁻⁶. 5 The maximum injection time in the orbital trap is 50 ms, the number of cycles is 5, the isolation window is 4.0 m / z, and the collision energy mode and energy values ​​are set to NCE: 15 (eV), NCE: 25 (eV), and NCE: 30 (eV) (to cover all collision energies in the target ion list). The minimum orbital trap capacity is 8 × 10⁻⁶. 3 The dynamic exclusion time is 10 seconds.

[0084] The established precise quality database of ethinylestradiol and its degradation products is shown in Table 2. The chromatogram and mass spectrum of ethinylestradiol are shown below. Figure 1 As shown, the chromatogram and mass spectrum of diacetylenol are as follows: Figure 2 As shown, the chromatogram and mass spectrum of β-estradiol 17-acetate are as follows: Figure 3 As shown, the chromatogram and mass spectrum of estriol are as follows: Figure 4 As shown, the chromatogram and mass spectrum of estrone are as follows: Figure 5 As shown, the chromatogram and mass spectrum of estradiol are as follows: Figure 6 As shown, the chromatogram and mass spectrum of 2-hydroxyestrone are as follows: Figure 7 As shown, the chromatogram and mass spectrum of 16α-hydroxyestrone are as follows: Figure 8 As shown in the figure. In the chromatogram, the vertical axis "Relative Abundance" represents the relative abundance, and the horizontal axis "Time" represents the time. In the mass spectrum, the vertical axis "Relative Abundance" represents the relative abundance, and the horizontal axis "m / z" represents the mass-to-charge ratio.

[0085] Table 2

[0086]

[0087] (2) Samples: 15 mg / L ethinylestradiol was added to *Scenedesmus tetracauda* and cultured in BG11 medium to achieve a concentration of 5 × 10⁻⁶. 7The algal cells were kept at a constant density of 100 cells / mL, and aseptic operation was maintained throughout the process to obtain the cultured *Scenedesmus tetraculus* algal solution. The cultured *Scenedesmus tetraculus* algal solution was mixed with methanol at a volume ratio of 1:1, and the algal cells were broken by vortexing. Ethinyl estradiol and its degradation products were extracted with methanol, and then the algal cells were separated by solid-phase extraction to obtain ethinyl estradiol and its degradation products.

[0088] (3) Sample detection: The same chromatographic, mass spectrometry ion source and mass spectrometry detection conditions were used. The samples were detected by ultra-high pressure liquid chromatography-quadrupole-electrostatic field track trap high resolution mass spectrometer.

[0089] (4) Results analysis: The chromatograms and mass spectra of the samples are as follows. Figures 9-12 As shown, the sample detection results are compared and analyzed with the established precise mass database and mass spectrometry library (when the parent ion and daughter ion match the information in the precise mass database and mass spectrometry library, it is determined that the corresponding compound is detected in the sample). It can be seen that the sample contains 17-acetic acid β-estradiol, estrone, estradiol, and 16α-hydroxyestradiol.

[0090] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A qualitative analysis method for ethinylestradiol and its degradation products, characterized in that, Includes the following steps: Establish a precise mass database and mass spectrometry library for ethinylestradiol and its degradation products: Prepare standard solutions of ethinylestradiol and its degradation products, and use ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry to determine the precise mass database and mass spectrometry library for ethinylestradiol and its degradation products. Sample detection: Ethinyl estradiol was added to microalgae and then cultured in a culture medium. The algal solution was then mixed with a solvent, the algal cells were broken, and the algal cells were separated by solid-phase extraction to obtain the sample. The sample was detected by ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry. Results analysis: The detection results of the sample were compared and analyzed with the established precise mass database and mass spectrometry library. When the detection results matched the information in the precise mass database and mass spectrometry library, it was determined that the analyte ethinylestradiol and its degradation products were detected in the sample. The ethinylestradiol degradation products include diacetylenol, β-estradiol 17-acetate, estriol, estrone, estradiol, 2-hydroxyestradiol and 16α-hydroxyestradiol; The precise mass database includes the chromatographic retention times of each analyte ethinylestradiol and its degradation products, as well as the precise mass numbers of a precursor ion and the daughter ions with higher response intensity after applying different collision energies to the analyte ethinylestradiol and its degradation products; the mass spectrometry library includes secondary mass spectra generated after applying different collision energies to the analyte ethinylestradiol and its degradation products. The chromatographic conditions of the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometer are as follows: Chromatographic column: Shim-pack Scepter C18-120 2.1×100 mm, 1.9 μm; Mobile phase A is: 0.1 wt% formic acid aqueous solution; Mobile phase B is: LC-MS grade methanol; Column temperature: 35 ℃; Flow rate: 0.3 mL / min; Injection volume: 2 μL; Elution method: gradient elution; The gradient elution procedure is as follows: At 0 minutes, mobile phase A was 60% and mobile phase B was 40%. At 10.6 minutes, mobile phase A was 35% and mobile phase B was 65%. At 16.5 minutes, mobile phase A was 0% and mobile phase B was 100%. At 19.25 minutes, mobile phase A was 0%, and mobile phase B was 100%. At 19.35 minutes, mobile phase A was 60% and mobile phase B was 40%. After 23 minutes, mobile phase A was 60% and mobile phase B was 40%. The mass spectrometry ion source conditions for the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometer are as follows: Electrospray ionization source; Negative ion mode; Spray voltage: 3.5 kV; Heater temperature: 50 ℃; Sheath gas flow rate: 33 arb; Auxiliary gas flow rate: 15 arb; Scavenging flow rate: 2 arb; Ion lens voltage frequency: 55.

0.

2. The qualitative analysis method according to claim 1, characterized in that, The mass spectrometry detection conditions of the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometer are as follows: Scanning mode: Level 1 full scan / Data-dependent level 2 daughter ion scan; Scan range: 200-350 m / z; Level 1 full scan: resolution 70,000, maximum orbital trap capacity 3 × 10⁻⁶ 6 The maximum injection time of the orbital trap is 100ms; Data-dependent secondary daughter ion scanning: Target ion list enabled, with parameters matching those in the precise mass database; resolution 17500; maximum orbital trap capacity 1×10⁻⁶. 5 The maximum injection time of the orbital trap is 50 ms, the number of cycles is 5, the isolation window is 4.0 m / z, and the collision energy mode and energy value are set to NCE: 15 eV, NCE: 25 eV, and NCE: 30 eV. The minimum orbital trap capacity is 8 × 10⁻⁶. 3 The dynamic exclusion time is 10 seconds.

3. The qualitative analysis method according to claim 1, characterized in that, The specific process for establishing an accurate quality database of ethinylestradiol and its degradation products includes the following steps: Standard solutions of ethinylestradiol and its degradation products at a concentration of 1000 μg / L were prepared and directly injected into a high-resolution mass spectrometer (HPLC-quadrupole-electrostatic field orbital trap) with ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap. Analysis was performed in both positive and negative ion modes. The negative ion mode was selected as the scanning mode. The precise mass number of the parent ion of the corresponding analyte was determined, and the precise mass number of the daughter ion with the highest response intensity was selected as the preset daughter ion. The precise mass number of the parent ion and the preset daughter ion were input into the mass tracking module, and the collision energy modes CE and NCE were switched to optimize the best CE and NCE and monitor the preset daughter ion. The final daughter ion was confirmed to be the daughter ion in the precise mass database. In the above process, key mass spectrometry parameters such as electrospray voltage, ion source temperature, sheath gas pressure, and resolution were optimized respectively; A mixed standard solution of ethinylestradiol and its degradation products with a concentration of 1000 μg / L was prepared. The ultra-high pressure liquid chromatography separation conditions were optimized, and the chromatographic retention time of each compound was obtained by chromatographic separation. Establish a precise mass database: Input the name, molecular formula, Chemical Abstracts number, precise mass number of the parent ion, precise mass number of the daughter ion, chromatographic retention time, and optimal collision energy for each compound.

4. The qualitative analysis method according to claim 1, characterized in that, The specific process for establishing a mass spectrometry library of ethinylestradiol and its degradation products includes the following steps: Standard solutions of ethinylestradiol and its degradation products with a concentration of 1000 μg / L were prepared and analyzed by ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry. The target compounds were broken down to obtain secondary mass spectra of each compound. All secondary mass spectra were input and stored to obtain a mass spectral library of all the compounds to be tested.

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