A method for detecting steroid hormones in serum

Abstract

The present invention relates to a method for detecting steroid hormones in serum, comprising the following steps: (1) adding 0.1 to 20 mg of a magnetic solid phase extractant to each milliliter of a sample to be tested, mixing, magnetically separating, and removing liquid; adding a washing solution, mixing, magnetically separating, and removing liquid; adding an eluent for elution, mixing, magnetically separating, and collecting the eluent to obtain a test solution; (2) detecting the hormone content in the test solution by liquid chromatography and tandem mass spectrometry; wherein the hormone is selected from any one of testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone sulfate, and 17α-hydroxyprogesterone or a combination thereof; the magnetic solid phase extractant comprises a magnetic core ferrosoferric oxide, a middle layer structure silica wrapping the magnetic core, and an outer layer mesoporous structure, wherein the outer layer mesoporous structure is selected from any one of polystyrene and polystyrene divinylbenzene or a combination thereof. The present invention has the characteristics of good stability, high accuracy and sensitivity, strong specificity, high recovery rate, simple and controllable operation, fast analysis speed, etc., and can be used for simultaneous quantitative detection of multiple hormones in clinical practice.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a method for detecting steroid hormones in serum. Background Art

[0002] Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disorder that often develops during adolescence. Clinically, irregular menstruation is the primary symptom, often accompanied by excessive body hair, acne, obesity, and infertility. In addition to ultrasound findings of polycystic ovaries, elevated serum androgen levels are also crucial for diagnosis. Studies have shown that serum levels of five hormones, primarily testosterone (T), as well as androstenedione (A4), dihydrotestosterone (DHT), dehydroepiandrosterone sulfate (DHEAS), and 17α-hydroxyprogesterone (17-OHP), differ significantly between PCOS patients and healthy controls. Accurately measuring hormone levels is crucial for the clinical diagnosis of PCOS.

[0003] Currently, immunoassays are mainly used clinically to measure hormones. This method has the disadvantages of poor specificity, often leading to inaccurate results due to interference from factors such as heterophagic antibodies, and it is impossible to obtain results for multiple hormones in a single test. Many literature reports have reported that liquid chromatography-tandem mass spectrometry (LC-MS / MS) is used to detect serum levels of the sex hormone testosterone (T). However, due to the low hormone content in the human body, matrix interference is serious. Therefore, before the sample to be tested is analyzed, the sample must be pre-treated to enrich the analyte and eliminate matrix interference, thereby improving analytical accuracy and detection sensitivity. However, existing pre-treatment methods have complex steps, long processing time, low throughput, high reagent cost, high requirements for operators, and high consumables cost for online extraction. Therefore, it is urgent to develop a detection method that can simultaneously measure five steroid hormones in serum. Summary of the Invention

[0004] The object of the present invention is to provide a method for detecting steroid hormones in serum, comprising the following steps:

[0005] (1) Add 0.1-20 mg of magnetic solid phase extraction agent per milliliter of the sample to be tested, mix, perform magnetic separation, and remove the liquid; add eluent, mix, perform magnetic separation, and remove the liquid; add eluent for elution, mix, perform magnetic separation, and collect the eluent, which is the sample to be tested;

[0006] (2) using liquid chromatography and tandem mass spectrometry to detect the hormone content in the test fluid;

[0007] Wherein, the hormone is selected from any one of testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone sulfate, 17α-hydroxyprogesterone or a combination thereof;

[0008] The magnetic solid phase extractant comprises a magnetic core ferrosoferric oxide, a middle layer structure of silicon dioxide wrapping the magnetic core, and an outer layer mesoporous structure, wherein the outer layer mesoporous structure is selected from any one of polystyrene and polystyrene divinylbenzene or a combination thereof.

[0009] In a preferred technical solution of the present invention, the magnetic solid phase extractant is selected from any one of a water-oil balance magnetic solid phase extractant (HLB), a strong cation exchange magnetic solid phase extractant (MCX), a strong anion exchange magnetic solid phase extractant (MAX), a weak cation exchange magnetic solid phase extractant (WCX), and a weak anion exchange magnetic solid phase extractant (WAX), or a combination thereof.

[0010] According to a preferred technical solution of the present invention, the magnetic solid-phase extractant further comprises a functional group with selective adsorption properties bonded within the mesoporous structure.

[0011] In a preferred technical solution of the present invention, the functional group is selected from any one of a hydrophilic group, a strong cation exchange group (MCX), a strong anion exchange group (MAX), a weak cation exchange group (WCX), and a weak anion exchange group (WAX).

[0012] In a preferred technical solution of the present invention, the functional group is selected from any one of N-vinyl pyrrolidone, a sulfonic acid group, a quaternary amine group, a carboxylate group, and a secondary amine group.

[0013] According to a preferred technical solution of the present invention, 1 to 15 mg, more preferably 5 to 10 mg, of magnetic solid phase extraction agent is added to each milliliter of the sample to be tested.

[0014] According to a preferred technical solution of the present invention, the eluent is any one of methanol and n-hexane or a combination thereof.

[0015] In a preferred technical solution of the present invention, the mass volume ratio of the magnetic solid phase extraction agent to the eluent is 1:0.1-0.5 mg / mL, preferably 1:0.2-0.3 mg / mL.

[0016] According to a preferred technical solution of the present invention, the eluent is an acetonitrile-methanol mixed solution, and the volume ratio of acetonitrile to methanol in the mixed solution is 9:1.

[0017] In a preferred technical solution of the present invention, the mass volume ratio of the magnetic solid phase extraction agent to the eluent is 1:0.01-0.1 mg / mL, preferably 1:0.025-0.05 mg / mL.

[0018] According to a preferred technical solution of the present invention, the magnetic solid phase extractant is first activated to prepare a magnetic extractant suspension, which is then added to the sample solution to be tested.

[0019] According to a preferred technical solution of the present invention, the preparation method of the magnetic extractant suspension is as follows:

[0020] a. Take a magnetic solid phase extractant and add it to methanol or ethanol at a mass volume ratio of 1:1~10g / mL, and shake evenly;

[0021] b. Add methanol at a mass-to-volume ratio of 1:0.01-0.1 mg / mL, mix well, perform magnetic separation, and remove the solution;

[0022] c. Add 5% methanol aqueous solution at a mass volume ratio of 1:0.01-0.1 mg / mL, mix well, perform magnetic separation, and remove the solution to prepare a magnetic extractant suspension.

[0023] According to a preferred technical solution of the present invention, in step a, the mass volume ratio of the magnetic solid phase extractant to methanol or ethanol is 1:5-8 g / mL.

[0024] According to a preferred technical solution of the present invention, in step b, the mass volume ratio of the magnetic solid phase extraction agent to methanol is 1:0.05-0.008 mg / mL.

[0025] According to a preferred technical solution of the present invention, in step c, the mass volume ratio of the magnetic solid phase extraction agent to the 5% methanol aqueous solution is 1:0.05-0.08 mg / mL.

[0026] According to a preferred technical solution of the present invention, the mixing method is ultrasonic mixing, with an ultrasonic frequency of 40-60 Hz, an ultrasonic power of 60-240 W, an ultrasonic temperature of 20-30 degrees, and an ultrasonic mixing time of 30 to 60 seconds.

[0027] In a preferred technical solution of the present invention, the magnetic separation method is to use a neodymium magnet to adsorb a magnetic solid phase extractant for separation.

[0028] According to a preferred technical solution of the present invention, the method for preparing the sample solution to be tested is to add a mixed internal standard solution to the sample to be tested, dilute with water, and vortex mix uniformly to obtain the sample solution to be tested.

[0029] According to a preferred technical solution of the present invention, the mixed internal standard solution contains a testosterone internal standard (T-d3), an androstenedione internal standard (A4-13C3), a dihydrotestosterone internal standard (DHT-d3), a dehydroepiandrosterone sulfate internal standard (DHEAS-d5), and a 17α-hydroxyprogesterone internal standard (17-OHP-d8); preferably, the concentrations of the testosterone internal standard (T-d3), the androstenedione internal standard (A4-13C3), the dihydrotestosterone internal standard (DHT-d3), and the 17α-hydroxyprogesterone internal standard (17-OHP-d8) in the mixed internal standard solution are all 10 ng / mL, and the concentration of the dehydroepiandrosterone sulfate internal standard (DHEAS-d5) is 1200 ng / mL.

[0030] According to a preferred technical solution of the present invention, the sample to be tested is serum obtained after coagulation treatment of the subject's venous blood or a related extract.

[0031] According to a preferred technical solution of the present invention, the liquid chromatography and tandem mass spectrometry detection method is to separate the extracted hormones by liquid chromatography and detect them by triple quadrupole tandem mass spectrometry.

[0032] In the preferred technical solution of the present invention, the chromatographic column is selected from a reversed-phase C18 chromatographic column, an electrophilic group-modified reversed-phase C18 chromatographic column, a phenyl-modified reversed-phase C18 chromatographic column, and a perfluorophenyl-modified reversed-phase C18 chromatographic column.

[0033] In a preferred technical solution of the present invention, the diameter of the chromatographic column is 3-5 mm, the length of the chromatographic column is 20-300 mm, and the particle size of the filler in the chromatographic column is 2-5 μm.

[0034] According to a preferred technical solution of the present invention, the chromatographic column model of the liquid chromatography is Phenomenex Kinetex C18 (2.6um, 2.1x50mm).

[0035] According to a preferred technical solution of the present invention, the mobile phases of the liquid chromatography are mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution containing 1-5 mmol / L ammonium fluoride; and mobile phase B is a methanol aqueous solution containing 1-5 mmol / L ammonium fluoride.

[0036] According to a preferred technical solution of the present invention, the flow rate of the liquid chromatography is 0.1-1 mL / min, preferably 0.4-0.8 mL / min.

[0037] According to a preferred technical solution of the present invention, the column temperature of the liquid chromatography is 30-40°C, preferably 35-37°C.

[0038] According to a preferred technical solution of the present invention, the injection volume of the liquid chromatography is 1-10 μL, preferably 2-5 μL.

[0039] The preferred technical solution of the present invention is that the mobile phase elution procedure is:

[0040] Time (min) A％ B％ 0 70 30 0.5 50 50 2 50 50 3.5 10 90 4.5 10 90 5 70 30 6 70 30

[0041] According to a preferred technical solution of the present invention, the tandem mass spectrometry conditions are as follows: ion source part: nebulizing gas 50 psi; heating auxiliary gas 55 psi; curtain gas 25 psi; ionization voltage 5500 V / -4500 V.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The method of the present invention enriches five hormones in serum through dispersed solid-phase extraction technology, and combines liquid chromatography-tandem mass spectrometry (LC-MS / MS) to analyze trace hormones in serum, which solves the problems of the existing technology that it is difficult to accurately determine the content of hormones in human serum, and most of them have long analysis time, complex sample pretreatment, few types of hormones to be measured, and cannot simultaneously measure the content of multiple hormones.

[0044] 2. The method of the present invention has the characteristics of good stability, high accuracy and sensitivity, strong specificity, high recovery rate, simple and controllable operation, and fast analysis speed. It can be used for the simultaneous quantitative detection of multiple androgens in clinical practice.

[0045] Unless otherwise stated, in this article,

[0046] T refers to testosterone;

[0047] A4 refers to androstenedione;

[0048] DHT refers to dihydrotestosterone;

[0049] DHEAS refers to dehydroepiandrosterone sulfate;

[0050] 17-OHP refers to 17α-hydroxyprogesterone.

[0051] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the MRM chromatogram of hormones in serum from Example 1; DETAILED DESCRIPTION

[0053] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0054] Example 1 Detection of hormones in serum

[0055] 1. Preparation of standard samples

[0056] The original standards of the five hormones were each prepared as a 1000 μg / mL methanol solution. The single-standard solutions of the five sex hormones were diluted in methanol to a concentration of 10 μg / mL. These single-standard solutions were mixed in methanol to create a mixed standard solution with concentrations of 10 ng / mL for T, A4, DHT, and 17-OHP, and 1200 ng / mL for DHEAS. Using blank serum (or a 4-5% aqueous solution of bovine serum albumin) devoid of the five sex hormones, these mixed standard solutions were serially diluted to create a series of standard curve working solutions with concentrations of 0.05, 0.1, 0.5, 1, 2, 5, and 10 ng / mL for T, A4, DHT, and 17-OHP, and 6, 12, 60, 120, 240, 600, and 1200 ng / mL for DHEAS. These solutions were then subjected to magnetic extraction.

[0057] The original isotope-labeled standards for the five sex hormones were prepared at a concentration of 1000 μg / mL in methanol. These were serially diluted and added to the working solutions at each point of the standard curve until the concentrations reached 0.5 ng / mL for T-d3, A4-13C3, DHT-d3, and 17-OHP-d8, and 60 ng / mL for DHEAS-d5.

[0058] 2. Pretreatment of samples to be tested

[0059] S1. Accurately weigh 0.5 g of Bonnacats-MA HLB magnetic extractant into a 5 mL glass vial and add 5 mL of ethanol. Shake thoroughly before use until all solids are suspended in the ethanol and no sediment remains at the bottom of the vial. This will yield a magnetic extractant suspension.

[0060] S2. Activate. Pipette 200 μL of methanol into the test tube and mix thoroughly with ultrasonic technology for 30 seconds. Place a rubidium magnet on the outside of the test tube near the bottom and let it sit for 30 seconds. Remove as much methanol as possible and discard.

[0061] S3. Balance the tube. Pipette 400 μL of 5% methanol-water solution into the test tube and mix thoroughly with ultrasonic waves for 30 seconds. Place a rubidium magnet on the outside of the test tube near the bottom and let it sit for 30 seconds. Pipette as much of the 5% methanol-water solution as possible and discard.

[0062] S4. Take 400 μL of serum, add 20 μL of a mixed internal standard solution of T-d3, A4-13C3, DHT-d3, 17-OHP-d8 at a concentration of 10 ng / mL, and DHEAS-d5 at a concentration of 1200 ng / mL, add 400 μL of water, and vortex to mix evenly to obtain the sample solution to be purified.

[0063] S5. Load the sample. Pipette 800 μL of the sample solution to be purified into the test tube and mix thoroughly using ultrasonic technology for 60 seconds. Place a rubidium magnet on the outside of the test tube near the bottom and let it sit for 30 seconds. Pipette as much of the sample solution as possible and discard.

[0064] In step S6, rinse 1, pipette 800 μL of 5% methanol in water into the test tube and mix thoroughly with ultrasound for 30 seconds. Place a rubidium magnet on the outside of the test tube near the bottom and let it sit for 30 seconds. Pipette as much of the 5% methanol in water as possible and discard.

[0065] In S7, Wash 2, pipette 400 μL of n-hexane into the test tube and mix thoroughly using ultrasonic technology for 30 seconds. Place a rubidium magnet on the outside of the test tube near the bottom and let it sit for 30 seconds. Remove as much n-hexane as possible and discard.

[0066] S8. Elution: Pipette 100 μL of a 9:1 acetonitrile / methanol mixture (referred to as the eluent) into the test tube and mix thoroughly using ultrasonic technology for 60 seconds. Place a rubidium magnet on the outside of the test tube near the bottom and allow to stand for 30 seconds. Pipette as much of the eluent as possible and transfer it to a 1.5 mL glass vial or a well of a 96-well plate.

[0067] S9. Dilution: add 200 μL of water to the eluate and vortex to mix evenly to obtain the eluate dilution solution.

[0068] 3. Analysis and testing of samples to be tested

[0069] The eluate dilution obtained in step S9 is analyzed and tested by liquid chromatography separation and triple quadrupole tandem mass spectrometry detection to obtain a sample spectrum.

[0070] Liquid chromatography conditions are as follows:

[0071] Column: Phenomenex Kinetex C18 2.6 μm, 2.1 x 50 mm

[0072] Flow rate: 0.4 mL / min

[0073] Column temperature: 35°C

[0074] Injection volume: 5 μL

[0075] Mobile phase: A, aqueous solution containing 5 mmol / L ammonium fluoride; B, methanol aqueous solution containing 5 mmol / L ammonium fluoride

[0076] Mobile phase elution program:

[0077] Time (min) A％ B％ 0 70 30 0.5 50 50 2 50 50 3.5 10 90 4.5 10 90 5 70 30 6 70 30

[0078] Tandem mass spectrometry conditions were as follows:

[0079] Ion source part: nebulizer gas 50psi; heating auxiliary gas 55psi; curtain gas 25psi; ionization voltage 5500V / -4500V.

[0080] Mass analyzer part:

[0081] Table 1 Hormone MRM mass spectrometry parameters

[0082]

[0083]

[0084] 4. Quantitative calculation results analysis: test results are shown in Figure 1 .

[0085] Example 1 Methodology Verification Results:

[0086] 1. Linearity and range

[0087] The hormone DHEAS was in the range of 20 to 5000 ng / mL, and the hormones 17-OHP, A4, DHT, and T were in the range of 0.02 to 10 ng / mL. The linear relationship was good, and the correlation coefficients were all greater than 0.998.

[0088] 2. Limit of Quantitation

[0089] The limit of quantification of the hormone DHEAS is 20 ng / mL, and the limit of quantification of the hormones 17-OHP, A4, DHT, and T is 0.02 ng / mL.

[0090] 3. Matrix Effect

[0091] The actual response values ​​A and standard response values ​​B of the five hormones were measured respectively. The matrix influence factors of the five hormones, that is, the response value ratio A / B, were all between 82.4% and 108%, meeting the validation requirements.

[0092] 4. Accuracy

[0093] The test was repeated three times using the method of the present invention. The recovery rates of the five hormones were all between 88.7% and 106%, and the RSD (n=3) was ≤8.1%, meeting the verification requirements.

[0094] 5. Precision

[0095] Blank plasma was spiked with appropriate amounts of five hormone reference standards to prepare precision test samples at low, medium, and high concentrations (DHEAS concentrations of 20, 50, and 1000 ng / mL; 17-OHP, A4, DHT, and T concentrations of 0.02, 0.05, and 1.0 ng / mL, respectively). These samples were assayed in two batches using the method described in this invention. The intra- and inter-batch relative standard deviations for the five hormones were all ≤8.7%, and the recoveries ranged from 88.7% to 107%, meeting validation requirements.

[0096] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting steroid hormones in serum, characterized in that: The following steps are involved: (1) Add 0.1-20 mg of magnetic solid phase extraction agent to each milliliter of the sample to be tested, mix, perform magnetic separation, and remove the liquid; add eluent, mix, perform magnetic separation, and remove the liquid; add eluent for elution, mix, perform magnetic separation, and collect the eluent, which is the liquid to be tested; the eluent is any one of methanol and n-hexane or a combination thereof; the eluent is a mixed solution of acetonitrile and methanol; The magnetic solid phase extractant is first activated to prepare a magnetic extractant suspension, which is then added to the sample solution to be tested. The preparation method of the magnetic extractant suspension is as follows: a. taking the magnetic solid phase extractant and adding it to methanol or ethanol at a mass volume ratio of 1:1 to 10 g / mL, and shaking it evenly; b. Add methanol at a mass volume ratio of 1:0.01-0.1 mg / mL, mix well, perform magnetic separation, and remove the solution; c. Add 5% methanol aqueous solution at a mass volume ratio of 1:0.01-0.1 mg / mL, mix well, perform magnetic separation, and remove the solution to prepare a magnetic extractant suspension; The magnetic solid-phase extractant is a water-oil balance magnetic solid-phase extractant HLB, comprising a magnetic core of ferrosoferric oxide, a middle layer of silica surrounding the magnetic core, and an outer layer of mesoporous structure, wherein the outer layer of mesoporous structure is selected from any one of polystyrene and polystyrene divinylbenzene, or a combination thereof; the magnetic solid-phase extractant further comprises a hydrophilic group with selective adsorption properties bonded to the mesoporous structure; (2) Detecting the hormone content in the test fluid using liquid chromatography and tandem mass spectrometry; wherein the steroid hormone is a combination of testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone sulfate and 17α-hydroxyprogesterone; The chromatographic column of the liquid chromatography is a reverse phase C18 chromatographic column, and the mobile phases of the liquid chromatography are mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution containing 1-5 mmol / L ammonium fluoride; mobile phase B is a methanol aqueous solution containing 1-5 mmol / L ammonium fluoride; The mobile phase elution procedure is: 。 2. The method according to claim 1, wherein The magnetic solid phase extraction agent is added in an amount of 1 to 15 mg per milliliter of the sample to be tested.

3. The method according to claim 2, wherein The magnetic solid phase extraction agent is added in an amount of 5-10 mg per milliliter of the sample to be tested.

4. The method according to claim 1, wherein The mass volume ratio of the magnetic solid phase extraction agent to the eluent is 1:0.1~0.5 mg / mL.

5. The method according to claim 4, wherein The mass volume ratio of the magnetic solid phase extraction agent to the eluent is 1:0.2-0.3 mg / mL.

6. The method according to claim 1, wherein The volume ratio of acetonitrile to methanol in the acetonitrile-methanol mixed solution is 9:

1.

7. The method according to claim 1, wherein The mass volume ratio of the magnetic solid phase extraction agent to the eluent is 1:0.01~0.1 mg / mL.

8. The method according to claim 7, wherein The mass volume ratio of the magnetic solid phase extraction agent to the eluent is 1:0.025-0.05 mg / mL.

9. The method according to claim 1, wherein In step a, the mass volume ratio of the magnetic solid phase extraction agent to ethanol is 1:5-8 g / mL.

10. The method according to claim 1, wherein In step b, the mass volume ratio of the magnetic solid phase extraction agent to methanol is 1:0.05-0.008 mg / mL.

11. The method according to claim 1, wherein In step c, the mass volume ratio of the magnetic solid phase extraction agent to 5% methanol aqueous solution is 1:0.05~0.08 mg / mL.

12. The method according to any one of claims 1 to 11, wherein: The mixing method is ultrasonic mixing, with an ultrasonic frequency of 40-60 Hz, an ultrasonic power of 60-240w, an ultrasonic temperature of 20-30 degrees, and an ultrasonic mixing time of 30-60s.

13. The method according to any one of claims 1 to 11, wherein: The magnetic separation method is to use a rubidium magnet to adsorb a magnetic solid phase extractant for separation.

14. The method according to any one of claims 1 to 11, wherein: The method for preparing the sample solution to be tested is to add the mixed internal standard solution to the sample to be tested, dilute with water, and vortex mix to obtain the sample solution to be tested.

15. The method according to claim 14, wherein The mixed internal standard solution contains testosterone internal standard T-d3, androstenedione internal standard A4-13C3, dihydrotestosterone internal standard DHT-d3, dehydroepiandrosterone sulfate internal standard DHEAS-d5 and 17α-hydroxyprogesterone internal standard 17-OHP-d8.

16. The method according to claim 15, wherein The concentrations of testosterone internal standard T-d3, androstenedione internal standard A4-13C3, dihydrotestosterone internal standard DHT-d3, and 17α-hydroxyprogesterone internal standard 17-OHP-d8 in the mixed internal standard solution are all 10 ng / mL, and the concentration of dehydroepiandrosterone sulfate internal standard DHEAS-d5 is 1200 ng / mL.

17. The method according to any one of claims 1 to 11, wherein: The liquid chromatography and tandem mass spectrometry detection method is to separate the extracted hormones by liquid chromatography and detect them by triple quadrupole tandem mass spectrometry.

18. The method according to claim 1, wherein The diameter of the chromatographic column is 3-5 mm, the length of the chromatographic column is 20-300 mm, and the particle size of the filler in the chromatographic column is 2-5 μm.

19. The method according to claim 18, wherein The chromatographic column model of the liquid chromatography is Phenomenex Kinetex C18, 2.6um, 2.1x50mm.

20. The method of claim 1, wherein The flow rate of the liquid chromatography is 0.1-1 mL / min.

21. The method according to claim 20, wherein The flow rate of the liquid chromatography was 0.4-0.8 mL / min.

22. The method of claim 1, wherein The column temperature of the liquid chromatography is 30-40°C.

23. The method according to claim 22, wherein The column temperature of the liquid chromatography is 35-37°C.

24. The method of claim 1, wherein The injection volume of the liquid chromatography was 1-10 μL.

25. The method of claim 24, wherein: The injection volume of the liquid chromatography was 2-5 μL.

26. The method according to any one of claims 1 to 11, wherein The tandem mass spectrometry conditions are as follows: ion source: nebulizer gas 50 psi; heating auxiliary gas 55 psi; curtain gas 25 psi; ionization voltage 5500 V / -4500 V.

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