Androgen back door pathway hormone detection kit, detection method and application

Abstract

The invention relates to an androgen back door pathway hormone detection kit, a detection method and application. The detection method comprises the following steps: pretreating a to-be-detected sample, and then carrying out liquid chromatography-tandem mass spectrometry detection; the pretreatment comprises the following steps: mixing the to-be-detected sample with an internal standard, adding an extracting agent, centrifuging, taking supernate, drying, and redissolving by using a redissolving agent; the extracting agent comprises a mixed solution of ethyl acetate and n-hexane; the resolvent comprises a methanol aqueous solution. The kit and the LC-MS / MS detection method for simultaneously detecting 5 alpha-dihydroprogesterone, 17 hydroxyl-dihydroprogesterone, 17 hydroxyl-tetrahydroprogesterone, androsterone, androstanediol and dihydrotestosterone are designed, rapid and accurate detection is achieved, high sensitivity is achieved, the kit and the method can be effectively applied to auxiliary diagnosis of 17 alpha-hydroxylase deficiency diseases, especially atypical 17 alpha-hydroxylase deficiency diseases and other diseases, and the clinical application prospect is wide. The method has high practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and relates to a kit, a detection method and an application for detecting hormones in the androgen backdoor pathway. Background Art

[0002] The androgen backdoor pathway is an alternative pathway that bypasses the traditional classical pathway during androgen biosynthesis. In the classical pathway, androgen synthesis mainly occurs in the testis, where cholesterol is converted into testosterone and dihydrotestosterone (DHT) through a series of enzymatic reactions. However, the backdoor pathway bypasses the testis and mainly occurs in the placenta, liver, and adrenal glands, where androgens are synthesized through different enzymes and substrates. It is an androgen metabolic pathway starting from progesterone and 17-hydroxyprogesterone, which automatically closes after the fetus is born. Those in whom this pathway fails to close automatically will exhibit various clinical manifestations related to abnormal adrenal cortex metabolism after birth, such as abnormal regulation of blood pressure, blood sugar, and blood lipids, and abnormal development of secondary sexual characteristics. Adult women often suffer from hyperandrogenism, amenorrhea, infertility, etc.; men may have gynecomastia, etc.

[0003] Related hormones in the androgen backdoor pathway include 5α-dihydroprogesterone (5α-DHP), 17-hydroxy-dihydroprogesterone (17OH-DHP), 17-hydroxy-allotetrahydroprogesterone (17OH-Allo), androsterone (Andr), 3α-diol, and dihydrotestosterone (DHT), etc., which can be used as disease diagnostic markers for clinical diagnosis. For example, 3α-diol can assist in differentiating the causes of hyperandrogenism, and dihydrotestosterone is used for the diagnosis of non-hyperandrogenic polycystic ovary syndrome, etc. However, the concentration of each hormone is at the pg / mL level. Therefore, various conventional techniques in medical laboratories, such as immunological methods and biochemical methods, cannot accurately identify each hormone structure and accurately measure it. Usually, ultra-high performance liquid chromatography-tandem mass spectrometry technology is required for detection. For example, CN115932084A discloses a method, a kit, and an application for detecting 5 steroid hormones in blood samples by ultra-high performance liquid chromatography-tandem mass spectrometry technology. The steroid hormones include testosterone, androstenedione, dihydrotestosterone, 17α-hydroxyprogesterone, and dehydroepiandrosterone sulfate. CN118858479A discloses a kit, a detection method, and a sample pretreatment method for simultaneously detecting 90 steroid hormones and their metabolites in urine samples. The 90 steroid hormones and their metabolites are estrone, 17α-estradiol, 5α-dihydroprogesterone, 17-carboxyprogesterone, etc. However, there are few reports on the detection of specific hormones such as 17-hydroxy-dihydroprogesterone, and there is also a lack of mature related detection kits on the market.

[0004] In summary, there is an urgent need to develop kits and detection methods with high sensitivity and specificity for detecting related hormones in the androgen backdoor pathway. Summary of the Invention

[0005] In view of the deficiencies of the prior art and the actual needs, the present invention provides a detection kit, detection method and application for hormones in the androgen backdoor pathway, in order to achieve the simultaneous detection of 6 specific hormones in the androgen backdoor pathway.

[0006] To achieve this purpose, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a detection method for hormones in the androgen backdoor pathway. The detection method includes: performing liquid chromatography-tandem mass spectrometry detection after pretreatment of the sample to be tested; the pretreatment includes: mixing the sample to be tested with an internal standard, adding an extraction agent, centrifuging to obtain the supernatant, drying and then redissolving with a reconstitution solvent to obtain a test solution; the extraction agent includes a mixed solution of ethyl acetate and n-hexane; the reconstitution solvent includes an aqueous methanol solution; the hormones in the androgen backdoor pathway include 5α-dihydroprogesterone (5α-DHP), 17-hydroxy-dihydroprogesterone (also known as allopregnan-3,20-dione-17α-ol, 17OH-DHP), 17-hydroxy-tetrahydroprogesterone (also known as 5α-pregnane-3α,17-diol-20-one, 17OH-Allo), androsterone (Andr), androstane diol (3α-diol) and dihydrotestosterone (DHT); the internal standard is a standard of the isotope-labeled hormone in the androgen backdoor pathway.

[0008] In the present invention, in view of the need to simultaneously detect 6 specific hormones in the androgen backdoor pathway, a targeted detection method is designed, including designing specific extraction agents and reconstitution solvents, etc. Overall, it can achieve the simultaneous qualitative and quantitative detection of 6 specific hormones in the androgen backdoor pathway, with high sensitivity and high practical application value.

[0009] It can be understood that the detection method of the present invention can be used for assisting clinical diagnosis and also for basic research for non-disease diagnosis purposes.

[0010] Preferably, the liquid chromatography-tandem mass spectrometry includes liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0011] Preferably, the volume ratio of ethyl acetate to n-hexane in the mixed solution of ethyl acetate and n-hexane is 1:(0.25 - 4), for example, it can be 1:1, 1:2, 1:3 or 1:4, etc., and preferably 1:(1 - 4).

[0012] In the present invention, for the detection of 6 specific hormones in the androgen backdoor pathway, a specific extraction agent is designed, which can further improve the response of the substance to be tested.

[0013] Preferably, the aqueous methanol solution comprises 30% - 60% aqueous methanol solution, for example, it can be 30%, 40%, 50% or 60%, etc., and preferably 40% - 50%.

[0014] In the present invention, for the detection of 6 specific androgen backdoor pathway hormones, a specific complex solution is designed, which can further improve the response of the substance to be detected.

[0015] Preferably, the sample to be detected comprises a blood sample of a subject, including any one of whole blood, plasma or serum.

[0016] Preferably, the liquid chromatography conditions include: gradient elution is adopted, mobile phase A comprises an aqueous solution containing 0.05% - 0.5% formic acid (for example, it can be 0.06%, 0.08%, 0.1%, 0.2%, 0.3% or 0.4%, etc., and preferably 0.08% - 0.12%); mobile phase B comprises a methanol solution; the flow rate of the mobile phase is 0.3 - 0.6 mL / min; the column temperature is 40 - 50 °C; the injection volume is 5 - 25 μL.

[0017] In the present invention, for the detection of 6 specific androgen backdoor pathway hormones, a specific mobile phase is designed, which can further improve the response of the substance to be detected.

[0018] Preferably, the gradient elution method includes: from 0 to 0.5 min, 30% - 60% mobile phase B; from 0.5 to 2 min, it rises to 55% - 75% mobile phase B; from 2 to 4.5 min, it rises to 70% - 90% mobile phase B; from 4.6 to 5.5 min, it maintains 80% - 95% mobile phase B; from 5.5 to 6.2 min, it drops to 30% - 60% mobile phase B.

[0019] Preferably, the chromatographic column of the liquid chromatography comprises a C18 column.

[0020] Preferably, the mass spectrometry conditions include: the ion source is ESI +, the capillary voltage is 1.0 kV - 4.0 kV, the cone voltage is 20 V - 50 V, the desolvation gas temperature is 450 °C - 550 °C, the desolvation gas flow rate is 1000 L / h - 1100 L / h, the cone gas flow rate is 130 L / h - 150 L / h; the nebulizer pressure is 6.0 bar - 7.0 bar.

[0021] Preferably, the mass spectrometry parameters are shown in Table 3.

[0022] In the present invention, for the detection of 6 specific androgen backdoor pathway hormones, specific mass spectrometry parameters are designed, which can further improve the detection effect.

[0023] Preferably, the detection method further includes the step of making a standard curve with a standard product, specifically including:

[0024] Prepare standard curve solutions with gradient concentrations using the reference standards of the hormones in the androgen backdoor pathway, and perform liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection on the standard curve solutions. Using the reference standard concentration as the abscissa and the peak area ratio of the target analyte to the internal standard as the ordinate, perform linear regression to obtain the standard curve.

[0025] Preferably, the LC-MS / MS detection uses isotope internal standard method for quantification.

[0026] Preferably, the isotope internal standard includes at least one of 5α-DHP-D8, 3α-diol-D3, or DHT-C3.

[0027] Preferably, the detection method further includes the step of performing quality control using two concentration levels of quality control samples, namely low and high concentrations.

[0028] Preferably, the preparation of the quality control samples includes: adding commercial hormone-free plasma into centrifuge tubes, and respectively adding appropriate amounts of reference standards to prepare low and high concentration quality control samples. The concentrations of 5α-DHP, 17OH-DHP, 17OH-Allo, Andr, 3α-diol, and DHT are 100 pg / mL, 100 pg / mL, 225 pg / mL, 100 pg / mL, 125 pg / mL, and 62.5 pg / mL respectively for the low concentration quality control; and the concentrations of 5α-DHP, 17OH-DHP, 17OH-Allo, Andr, 3α-diol, and DHT are 3000 pg / mL, 3000 pg / mL, 6750 pg / mL, 3000 pg / mL, 3750 pg / mL, and 1875 pg / mL respectively for the high concentration quality control.

[0029] In a second aspect, the present invention provides a kit for detecting hormones in the androgen backdoor pathway, which includes: reference standards, isotope internal standards, extractants, and reconstitution solvents.

[0030] Preferably, the reference standards include at least one of the reference standards of 5α-dihydroprogesterone, 17-hydroxy-dihydroprogesterone, 17-hydroxy-tetrahydroprogesterone, androsterone, androstane diol, or dihydrotestosterone; the isotope internal standards include at least one of 5α-DHP-D8, 3α-diol-D3, or DHT-C3;

[0031] Preferably, the extractant includes a mixture of ethyl acetate and n-hexane.

[0032] Preferably, the reconstitution solvent includes an aqueous methanol solution.

[0033] Preferably, the hormones in the androgen backdoor pathway include 5α-dihydroprogesterone, 17-hydroxy-dihydroprogesterone, 17-hydroxy-tetrahydroprogesterone, androsterone, androstane diol, and dihydrotestosterone.

[0034] Preferably, the method for using the kit includes the androgen backdoor pathway hormone detection method described in the first aspect.

[0035] In a third aspect, the present invention provides the use of the androgen backdoor pathway hormone detection method described in the first aspect or the androgen backdoor pathway hormone detection kit described in the second aspect in at least one of the following aspects:

[0036] (1) Application in the simultaneous detection of 5α-dihydroprogesterone, 17-hydroxy-dihydroprogesterone, 17-hydroxy-allopregnanolone, androsterone, androstanediol and dihydrotestosterone;

[0037] (2) Use in detecting at least one of 5α-dihydroprogesterone, 17-hydroxy-dihydroprogesterone, 17-hydroxy-allopregnanolone, androsterone, androstanediol and dihydrotestosterone;

[0038] (3) Application in the preparation of products for screening or assisting in the screening of endocrine diseases.

[0039] Preferably, the endocrine disease includes diseases such as 17α-hydroxylase deficiency.

[0040] Compared with the prior art, the present invention has at least the following beneficial effects:

[0041] The present invention designs a specific detection method for 5α-dihydroprogesterone, 17-hydroxydihydroprogesterone, 17-hydroxy-allosterone, androstenone, androstanediol and dihydrotestosterone, controls the synergy of multiple factors, realizes rapid and accurate detection of 6 androgen backdoor pathway hormones, has high specificity, sensitivity and accuracy, simplifies the operation steps, improves the detection efficiency, and is conducive to popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the detection spectrum of 5α-DHP;

[0043] Figure 2 This is the detection spectrum of 17OH-DHP;

[0044] Figure 3 It is the 17OH-Allo detection spectrum;

[0045] Figure 4 Detection spectrum for Andr;

[0046] Figure 5 This is the detection spectrum of 3α-diol;

[0047] Figure 6 It is the DHT detection spectrum;

[0048] Figure 7 This is the response result diagram of the substance to be tested using different extractants;

[0049] Figure 8 Response result graph for detecting the substance to be measured using different complex solvents. Detailed implementation mode

[0050] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation modes. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the claimed rights of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0051] For those not specifying specific technologies or conditions in the examples, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product instructions. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0052] In the specific embodiments of the present invention, the information of each standard product is shown in Table 1.

[0053] Table 1

[0054]

[0055]

[0056] Example 1

[0057] In this example, a kit for detecting hormones in the androgen backdoor pathway is prepared.

[0058] 1) Preparation of standard curve solution: Use an electronic balance with a precision of one in a hundred thousand to weigh 1 - 5 mg of 5α-DHP, 17OH-DHP, 17OH-Allo, Andr, 3α-diol, and DHT standard product powders into 2 mL brown injection vials respectively. Add 1 mL of methanol to each vial and vortex until the powder is completely dissolved to obtain single-standard stock solution 1. Take appropriate amounts of single-standard stock solution 1 into 2 mL brown injection vials respectively, add methanol to dilute to 0.1 mg / mL to obtain single-standard stock solution 2. Take appropriate amounts of single-standard stock solution 2 into 2 mL brown injection vials respectively, add appropriate methanol to obtain a mixed-standard stock solution containing 6 markers. Take appropriate amounts of the mixed-standard stock solution into centrifuge tubes and add appropriate 50% methanol to dilute to obtain the standard curve solution. The concentration ranges of 5α-DHP, 17OH-DHP, and Andr are 8 pg / mL - 4000 pg / mL; the concentration range of 17OH-Allo is 18 pg / mL - 9000 pg / mL; the concentration range of 3α-diol is 10 pg / mL - 5000 pg / mL; the concentration range of DHT is 5 pg / mL - 2500 pg / mL, as shown in Table 2 specifically;

[0059] Table 2

[0060]

[0061] 2) Preparation of mixed internal standard solution: Weigh 1 - 5 mg of isotope-labeled 5α-DHP-D8, 3α-diol-D3, and DHT-C3 (for the detection of 17OH-DHP, 17OH-Allo, and Andr, 3α-diol-D3 is still used as the internal standard) internal standard powder respectively using an electronic balance with a precision of one in a hundred thousand into 2 mL brown injection vials. Add 1 mL of methanol to each vial and vortex until the powder is completely dissolved to obtain a single internal standard stock solution 1. Take appropriate amounts of the single internal standard stock solution 1 into 2 mL brown injection vials, add methanol to dilute to 0.1 mg / mL to obtain a single internal standard stock solution 2. Take appropriate amounts of the single internal standard stock solution 2 into 2 mL brown injection vials, add an appropriate amount of methanol to obtain a mixed internal standard stock solution. Take an appropriate amount of the mixed internal standard stock solution into a centrifuge tube, add an appropriate amount of 50% methanol to dilute to obtain a mixed internal standard solution, with the concentration of 5α-DHP-D8 being 0.1 μg / mL, the concentration of 3α-diol-D3 being 0.7 μg / mL, and the concentration of DHT-C3 being 0.1 μg / mL.

[0062] 3) Preparation of extractant: Take ethyl acetate and n-hexane respectively into a volumetric flask, mix well by ultrasonic, and the volume ratio of the two is 1:1.

[0063] Preparation of re-solvent: Take water and methanol respectively into a volumetric flask, mix well by ultrasonic, and the volume ratio of the two is 1:1.

[0064] 4) Two concentration levels of quality control samples

[0065] Preparation of quality control samples: Take commercial hormone-free plasma and add it to a centrifuge tube. Add appropriate amounts of the mixed standard stock solution to prepare low and high concentration quality control samples. The concentrations of 5α-DHP, 17OH-DHP, 17OH-Allo, Andr, 3α-diol, and DHT are respectively: 100 pg / mL, 100 pg / mL, 225 pg / mL, 100 pg / mL, 125 pg / mL, 62.5 pg / mL for low concentration quality control; The concentrations of 5α-DHP, 17OH-DHP, 17OH-Allo, Andr, 3α-diol, and DHT are respectively: 3000 pg / mL, 3000 pg / mL, 6750 pg / mL, 3000 pg / mL, 3750 pg / mL, 1875 pg / mL for high concentration quality control.

[0066] Example 2

[0067] This example designs a method for detecting hormones in the androgen backdoor pathway.

[0068] (1) LC-MS / MS detection.

[0069] Sample pretreatment: Take 20 μL of the internal standard working solution into a centrifuge tube; add 500 μL of the standard curve solution / quality control / sample respectively, and mix well by shaking; add 1 mL of the extraction agent (ethyl acetate and n-hexane, 1:1) into the centrifuge tube, vortex for 5 min; centrifuge for 5 min, transfer the supernatant to a 96-well plate, dry by nitrogen blowing, then add 60 μL of the reconstitution solvent (methanol and water, 1:1), mix well by shaking, and perform on-machine detection.

[0070] Liquid phase conditions: The chromatographic column model is a C18 column, and the column temperature is 55 °C. Mobile phase A is an aqueous solution of 0.1% formic acid (FA); mobile phase B is methanol. Chromatographic separation of the markers is achieved by gradient elution. The gradient elution conditions are (mobile phase A + mobile phase B is 100%): 0 - 0.5 min, 55% mobile phase B, from 0.5 - 2 min it rises to 70% mobile phase B, from 2 - 4.5 min it rises to 85% mobile phase B, from 4.6 - 5.5 min it maintains 95% mobile phase B, from 5.5 - 6.2 min it drops to 55% mobile phase B; the flow rate is 0.3 mL / min; the injection volume is 20 μL.

[0071] Mass spectrometry conditions:

[0072] Mass spectrometry conditions: The ion source is ESI+; capillary voltage: 3.0 kV; desolvation temperature: 500 °C; desolvation gas flow rate: 1000 L / h; cone gas flow rate: 140 L / h; nebulizer pressure: 6.0 bar. The mass spectrometry parameters are shown in Table 3.

[0073] Table 3

[0074]

[0075] (2) Data analysis

[0076] 1) Plot the standard curve

[0077] Taking the concentration of the standard sample as the abscissa (x) and the ratio of the peak areas of the target analyte to the internal standard in the standard sample as the ordinate (y), 6 curves are fitted by TargetLynx software (internal standard method). Taking DHT as an example, the curve equation is y = 0.000960997x + 0.000525062.

[0078] 2) Calculate the contents of 5α-DHP, 17OH-DHP, 17OH-Allo, Andr, 3α-diol and DHT in the sample: After the sample is detected on the machine, the ratio of the peak areas of the target analyte to the internal standard in the sample (y) is obtained, and it is calculated by substituting into the standard curves of each index respectively. Taking DHT as an example, x = (y - 0.000525062) / 0.000960997

[0079] 3) Calculate accuracy, precision, and recovery rate

[0080] Accuracy: The accuracy of the method is evaluated by the spiked recovery rate. The calculation formula is: (measured concentration of the spiked sample - background concentration of the sample) / theoretical spiked concentration

[0081] Precision: Calculate the standard deviation and mean of each index for all samples in different batches. The calculation formula: CV = standard deviation / mean

[0082] Curve recovery rate: The calculation formula is: (measured value - theoretical value) / theoretical value

[0083] The detection includes the following steps:

[0084] 1) Take 300 μL of hormone-free plasma and add each standard sample. Add 20 μL of the mixed internal standard solution, vortex for 2 min, then let it stand in the dark for 5 min. Add 1 mL of the extractant (ethyl acetate and n-hexane, 1:1), vortex for 5 min, and centrifuge (4 °C, 12,000 rpm, 5 min). Take 600 μL of the supernatant and transfer it to a 96-well plate, dry it under nitrogen blowing, add 60 μL of the reconstitution solvent (methanol and water, 1:1), vortex for 2 min, and detect using LC-MS / MS. The molecular formulas and detection spectra of 5α-DHP, 17OH-DHP, 17OH-Allo, Andr, 3α-diol, and DHT are as Figures 1 - 6 shown

[0085] In addition, compare the effects of using different extractants and reconstitution solvents in the design. Replace the extractant with n-hexane, ethyl acetate:n-hexane = 2:8, ethyl acetate:n-hexane = 8:2, and ethyl acetate for detection respectively. The total amount of the extractant remains unchanged, and the other conditions are the same. The response results are as Figure 7 shown. Using a combination of ethyl acetate and n-hexane can improve the response of the analytes and is suitable for the simultaneous detection of 6 hormones. Replace the reconstitution solvent with 30% methanol, 40% methanol, and 60% methanol for detection respectively. The total amount of the reconstitution solvent remains unchanged, and the other conditions are the same. The response results are as Figure 8 shown. It can be seen that controlling the specific reconstitution solvent can further improve the response value of the analytes and is suitable for the simultaneous detection of 6 hormones

[0086] Example 3

[0087] This example analyzes and validates the hormone detection method for the androgen backdoor pathway

[0088] (1) Detection steps:

[0089] ① Take 20 μL of the internal standard working solution into a centrifuge tube

[0090] ② Add 500 μL of standard curve solution / quality control / sample respectively, and mix well by shaking.

[0091] ③ Add 1 mL of extractant (ethyl acetate and n - hexane, 1:1) into the centrifuge tube, and vortex for 5 min.

[0092] ④ Centrifuge for 5 min, transfer the supernatant to a 96 - well plate. After drying by nitrogen blowing, add 60 μL of re - solvent (methanol and water, 1:1), mix well by shaking, and perform on - machine detection (refer to the LC - MS / MS detection conditions in Example 2).

[0093] (2) Performance verification

[0094] 1) Limit of quantification (LOQ)

[0095] Prepare samples at 5 concentration levels, with 6 parallel samples for each concentration. Check the coefficient of variation (CV) and relative error (RE). It is required that CV < 20%, and RE is within ±20%. The LOQ concentration levels of each index are 5α - DHP 8 pg / mL, 17OH - DHP 8 pg / mL, 17OH - Allo 18 pg / mL, 3α - diol 9 pg / mL, Andr 8 pg / mL, DHT 4 pg / mL. This indicates that the detection method of the present invention has good sensitivity.

[0096] 2) Linearity

[0097] Continuously process 3 batches of standard curve solutions over 2 days, and check the linearity R of the fitting curves between batches 2 , calculate the mean value of curve points, recovery rate, and CV. It is required that linearity R 2 ≥0.995; the recovery rate is within ±20%, and for other concentrations within ±15%; the CV between batches < 20%. The recovery rate of each curve point of all indicators is between - 8% - 8%, all meeting the requirements; R 2 is 0.997 - 0.998, all > 0.995.

[0098] 3) Precision

[0099] Intra - batch precision: Use three concentration levels of low (L), medium (M), and high (H) (concentration requirements: within the concentration range of the normal reference population, 2 - 5 times the upper limit of the reference population concentration, more than 8 times the upper limit of the reference population concentration. See Table 4 for details), with 6 parallel samples. It is required that the CV of the low concentration < 20%, and the CV of the medium and high concentrations < 15%.

[0100] Inter - batch precision: Use three concentration levels of low, medium, and high, process 4 batches within 3 days, with 5 parallel samples for each batch. It is required that the CV of the low concentration < 20%, and the CV of the medium and high concentrations < 15%.

[0101] The within-batch precision CV of all indicators is between 1% and 4%; the between-batch precision CV is between 3% and 7%, all meeting the requirements.

[0102] Table 4

[0103]

[0104] 4) Accuracy

[0105] A spiked recovery experiment was carried out using hormone-free plasma as the blank matrix. The recovery rates were investigated at three concentration levels of low (L), medium (M), and high (H) (Table 4), and 3 parallel samples were processed. The low-concentration recovery rate was required to be within ±20%, and the medium- and high-concentration recovery rates were required to be within ±15%. The spiked recovery rates of all indicators at the three concentration levels of L, M, and H were between -14% and 11%, all meeting the requirements.

[0106] In summary, the present invention designs a kit for simultaneously detecting 5α-dihydroprogesterone, 17-hydroxy-dihydroprogesterone, 17-hydroxy-tetrahydroprogesterone, androsterone, androstane diol, and dihydrotestosterone, as well as an LC-MS / MS detection method, which realizes rapid and accurate detection, has high sensitivity, can be effectively applied to the auxiliary diagnosis of diseases such as 17α-hydroxylase deficiency, and has high practical application value.

[0107] The applicant declares that the above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for detecting hormones in the androgen backdoor pathway, characterized in that, The detection method includes: performing liquid chromatography-tandem mass spectrometry detection after pretreatment of the sample to be tested; The pretreatment includes: mixing the sample to be tested with an internal standard, adding an extraction agent, centrifuging to obtain the supernatant, drying and then redissolving with a reconstitution solvent; The extraction agent includes a mixture of ethyl acetate and n-hexane; The reconstitution solvent includes an aqueous methanol solution; The androsterone backdoor pathway hormones include 5α-dihydroprogesterone, 17-hydroxy-dihydroprogesterone, 17-hydroxy-tetrahydroprogesterone, androsterone, androstane diol, and dihydrotestosterone; The internal standard is a standard of the isotope-labeled androsterone backdoor pathway hormone.

2. The androgen backdoor pathway hormone detection method according to claim 1, wherein In the mixture of ethyl acetate and n-hexane, the volume ratio of ethyl acetate to n-hexane is 1:(0.25 - 4).

3. The androgen backdoor pathway hormone detection method according to claim 1 or 2, characterized in that, The aqueous methanol solution includes 30% - 60% aqueous methanol solution.

4. The androgen backdoor pathway hormone detection method according to any one of claims 1-3, characterized in that, The sample to be tested includes a blood sample of a subject, including any one of whole blood, plasma or serum.

5. The androgen backdoor pathway hormone detection method according to any one of claims 1-4, characterized in that, The liquid chromatography conditions include: gradient elution is adopted, mobile phase A includes an aqueous solution containing 0.05% - 0.5% formic acid; mobile phase B includes a methanol solution; the flow rate of the mobile phase is 0.3 - 0.6 mL / min; the column temperature is 40 - 50 °C; the injection volume is 5 - 25 μL; Preferably, the gradient elution method includes: from 0 to 0.5 min, 30% - 60% of mobile phase B, rising to 55% - 75% of mobile phase B from 0.5 to 2 min, rising to 70% - 90% of mobile phase B from 2 to 4.5 min, maintaining 80% - 95% of mobile phase B from 4.6 to 5.5 min, and dropping to 30% - 60% of mobile phase B from 5.5 to 6.2 min.

6. The androgen backdoor pathway hormone detection method according to any one of claims 1-5, characterized in that The mass spectrometry conditions include: the ion source is ESI+, the capillary voltage is 1.0 kV - 4.0 kV, the cone voltage is 20 V - 50 V, the desolvation gas temperature is 450 °C - 550 °C, the desolvation gas flow rate is 1000 L / h - 1100 L / h, the cone gas flow rate is 130 L / h - 150 L / h; the nebulizer pressure is 6.0 bar - 7.0 bar.

7. The androgen backdoor pathway hormone detection method according to any one of claims 1-6, characterized in that, The detection method further includes the step of making a standard curve with a standard product, specifically including: Preparing standard curve solutions with gradient concentrations using the standard of the androsterone backdoor pathway hormone, performing liquid chromatography-tandem mass spectrometry detection on the standard curve solutions, using the standard product concentration as the abscissa and the peak area ratio of the target analyte to the internal standard as the ordinate, and performing linear regression to obtain the standard curve.

8. The androgen backdoor pathway hormone detection method according to any one of claims 1-7, characterized in that, The liquid chromatography-tandem mass spectrometry detection uses the isotope internal standard method for quantification; Preferably, the isotope internal standard includes at least one of 5α-DHP-D8, 3α-diol-D3, or DHT-C3; Preferably, the detection method further includes the step of performing quality control using two concentration quality control products, low and high.

9. An androgen backdoor pathway hormone detection kit, characterized in that, The kit includes: a standard product, an isotope internal standard, an extraction agent, and a reconstitution solvent; The standard product includes at least one of the standards of 5α-dihydroprogesterone, 17-hydroxy-dihydroprogesterone, 17-hydroxy-tetrahydroprogesterone, androsterone, androstane diol, or dihydrotestosterone; the isotope internal standard includes at least one of 5α-DHP-D8, 3α-diol-D3, or DHT-C3; The extractant includes a mixed solution of ethyl acetate and n-hexane; The complex solvent includes an aqueous methanol solution; The androgen backdoor pathway hormones include 5α-dihydroprogesterone, 17-hydroxy-dihydroprogesterone, 17-hydroxy-tetrahydroprogesterone, androsterone, androstane diol, and dihydrotestosterone; The usage method of the kit includes the androgen backdoor pathway hormone detection method described in any one of claims 1-8.

10. Application of the androgen backdoor pathway hormone detection method described in any one of claims 1-8 or the androgen backdoor pathway hormone detection kit described in claim 9 in at least one of the following aspects: (1) Application in simultaneously detecting 5α-dihydroprogesterone, 17-hydroxy-dihydroprogesterone, 17-hydroxy-tetrahydroprogesterone, androsterone, androstane diol, and dihydrotestosterone; (2) Application in detecting at least one of 5α-dihydroprogesterone, 17-hydroxy-dihydroprogesterone, 17-hydroxy-tetrahydroprogesterone, androsterone, androstane diol, and dihydrotestosterone; (3) Application in preparing a product for screening or assisting in screening endocrine diseases.

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