Kit for simultaneously detecting six steroid hormones and application thereof

Abstract

The invention provides a kit for simultaneously detecting six steroid hormones and application of the kit. The steroid hormones comprise testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone and 17alpha-hydroxyprogesterone. The kit comprises extract liquor, a mobile phase additive and a standard quality control product. The kit provided by the invention can be used for simultaneously detecting testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone and 17alpha-hydroxyprogesterone, and has the advantages of high accuracy, low quantitation limit and wide detection range.

Description

Technical Field

[0001] The invention belongs to the technical field of clinical detection, and specifically relates to a kit for simultaneously detecting six types of steroid hormones and an application thereof. Background Art

[0002] Polycystic ovary syndrome (PCOS) is a common endocrine disease in women of childbearing age and a major cause of female infertility. Since a large proportion of PCOS patients suffer from hyperandrogenism, the main androgens involved are testosterone (T), 4-androstenedione (AD), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS) and dihydrotestosterone (DHT), and most PCOS patients have elevated levels of two or more androgens. The "Expert Consensus on Androgen Mass Spectrometry Detection in Polycystic Ovary Syndrome" published in 2023 recommends accurate determination of androgens such as T, A4, DHEA / DHEAS and DHT; and the measurement of 17α-hydroxyprogesterone (17-OHP) is the main means of diagnosing congenital adrenal hyperplasia and Cushing's syndrome caused by 21-hydroxylase deficiency. At present, the pretreatment methods for the above six compounds include liquid-liquid extraction, solid phase extraction, etc., and the detection methods include chemiluminescence immunoassay, LC-MS / MS, etc. However, due to the large difference in the concentration ranges of the six compounds and the large difference in polarity between DHEAS and the other five hormones, conventional pretreatment methods are usually difficult to take into account the co-detection of DHEAS and the other hormones. When protein precipitation is used, DHEAS is prone to overload due to its high concentration, and diluting the sample will cause the sensitivity of the other compounds to decrease. Therefore, the dynamic linear range that DHEAS can provide is narrow, and it needs to be re-diluted when it exceeds the upper limit of detection. When liquid-liquid extraction is used, DHEAS has a large polarity and the extraction solvent is incompatible with the other compounds. The extraction rate and repeatability are relatively poor when extracted in human samples; at the same time, DHT has a weak signal response and the concentration in normal human plasma is less than 0.3ng / mL, so its sensitivity is usually a difficulty in research and development. The mainstream test kits currently on the market usually select one between DHEA and DHEAS, and do not support the simultaneous detection of both. There is usually no detection indicator for DHT, or the sensitivity of DHT is not enough to support clinical application, which brings great difficulties to clinical diagnosis.

[0003] Therefore, how to provide a method that can accurately and simultaneously detect the above six steroid hormones has become an urgent problem to be solved. Summary of the invention

[0004] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kit for simultaneously detecting six steroid hormones and its application. The kit provided by the present invention can be used to simultaneously detect testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone and 17α-hydroxyprogesterone, and has the advantages of high accuracy, low quantification limit and wide detection range.

[0005] To achieve the purpose of this invention, the following technical solutions are adopted:

[0006] In a first aspect, the present invention provides a kit for simultaneously detecting six steroid hormones, and the steroid hormones include testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone and 17α-hydroxyprogesterone;

[0007] The kit includes an extraction solution, a mobile phase additive and a standard quality control product;

[0008] The extraction solution includes an extractant, a precipitant and a diluent. The extractant includes ethyl acetate (EA) and / or n-hexane (Hex), the precipitant includes alcohols and / or acetonitrile, and the diluent includes an aqueous solution of alcohols and / or acetonitrile;

[0009] The mobile phase additive includes mobile phase additive A, and the mobile phase additive A is an aqueous ammonium fluoride solution or an ammonium fluoride methanol solution;

[0010] The standard quality control product includes a standard curve solution, a quality control reagent, an internal standard and a blank matrix;

[0011] The standard curve solution includes standard product solutions of testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone and 17α-hydroxyprogesterone with at least 5 concentration gradients;

[0012] The quality control reagent includes a low-level quality control reagent and a high-level quality control reagent, and the low-level quality control reagent and the high-level quality control reagent are standard product solutions of testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone and 17α-hydroxyprogesterone;

[0013] The internal standard is an isotope internal standard of the steroid hormone;

[0014] The blank matrix is a substitute matrix.

[0015] The above-mentioned specific kit is used to simultaneously detect testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone and 17α-hydroxyprogesterone, and has the advantages of high accuracy and low quantification limit.

[0016] Preferably, the extractant is ethyl acetate and n-hexane, and the volume ratio of ethyl acetate to n-hexane is 1:(3 - 5), such as 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, etc., but not limited to the values listed above. Other unlisted values within the above value range are equally applicable.

[0017] The selection of the specific extractant and the control of the volume ratio can effectively reduce the baseline noise and improve the detection accuracy.

[0018] Preferably, the alcohols include methanol and / or ethanol.

[0019] The mobile phase additive further includes mobile phase additive B, which is an aqueous ammonium fluoride solution or an ammonium fluoride methanol solution, and the components of mobile phase additive A and mobile phase additive B are different.

[0020] Preferably, the concentration of the aqueous ammonium fluoride solution is 25 - 250 mM, such as 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM or 250 mM, etc., but not limited to the values listed above. Other unlisted values within the above value range are equally applicable.

[0021] Preferably, the concentration of the ammonium fluoride methanol solution is 25 - 250 mM, such as 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM or 250 mM, etc., but not limited to the values listed above. Other unlisted values within the above value range are equally applicable.

[0022] Preferably, in the standard curve solution, the concentration of the testosterone standard solution is 0.25 - 1000 ng / mL, the concentration of the androstenedione standard solution is 0.1 - 400 ng / mL, the concentration of the dihydrotestosterone standard solution is 0.5 - 2000 ng / mL, the concentration of the 17α-hydroxyprogesterone standard solution is 0.5 - 2000 ng / mL, the concentration of the dehydroepiandrosterone sulfate standard solution is 100 - 400000 ng / mL, and the concentration of the dehydroepiandrosterone standard solution is 12.5 - 50000 ng / mL; wherein the concentration of the testosterone standard solution can be 0.25 ng / mL, 1 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL or 1000 ng / mL, etc., the concentration of the androstenedione standard solution can be 0.1 ng / mL, 0.4 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 350 ng / mL or 400 ng / mL, etc., the concentration of the dihydrotestosterone standard solution can be 0.5 ng / mL, 2 ng / mL, 200 ng / mL, 400 ng / mL, 600 ng / mL, 800 ng / mL, 1000 ng / mL, 1200 ng / mL, 1400 ng / mL, 1600 ng / mL, 1800 ng / mL or 2000 ng / mL, etc., the concentration of the 17α-hydroxyprogesterone standard solution can be 0.5 ng / mL, 2 ng / mL, 200 ng / mL, 400 ng / mL, 600 ng / mL, 800 ng / mL, 1000 ng / mL, 1200 ng / mL, 1400 ng / mL, 1600 ng / mL, 1800 ng / mL or 2000 ng / mL, etc., the concentration of the dehydroepiandrosterone sulfate standard solution can be 100 ng / mL, 400 ng / mL, 50000 ng / mL, 100000 ng / mL, 150000 ng / mL, 200000 ng / mL, 250000 ng / mL, 300000 ng / mL, 350000 ng / mL or 400000 ng / mL, etc., and the concentration of the dehydroepiandrosterone standard solution can be 12.5 ng / mL, 50 ng / mL, 5000 ng / mL, 10000 ng / mL, 15000 ng / mL, 20000 ng / mL, 25000 ng / mL, 30000 ng / mL, 35000 ng / mL, 40000 ng / mL, 45000 ng / mL or 50000 ng / mL, etc., but is not limited to the values listed above, and other unlisted values within the above numerical ranges are equally applicable.

[0023] Preferably, in the low-level quality control reagent, the concentration of the standard solution of testosterone is 6-7 ng / mL, the concentration of the standard solution of androstenedione is 2-3 ng / mL, the concentration of the standard solution of dihydrotestosterone is 10-15 ng / mL, the concentration of the standard solution of 17α-hydroxyprogesterone is 10-15 ng / mL, the concentration of the standard solution of dehydroepiandrosterone sulfate is 2000-3000 ng / mL, and the concentration of the standard solution of dehydroepiandrosterone is 300-350 ng / mL; wherein the concentration of the standard solution of testosterone can be 6 ng / mL, 6.1 ng / mL, 6.2 ng / mL, 6.3 ng / mL, 6.4 ng / mL, 6.5 ng / mL, 6.6 ng / mL, 6.7 ng / mL, 6.8 ng / mL, 6.9 ng / mL or 7 ng / mL, etc., the concentration of the standard solution of androstenedione can be 2 ng / mL, 2.1 ng / mL, 2.2 ng / mL, 2.3 ng / mL, 2.4 ng / mL, 2.5 ng / mL, 2.6 ng / mL, 2.7 ng / mL, 2.8 ng / mL, 2.9 ng / mL or 3 ng / mL, etc., the concentration of the standard solution of dihydrotestosterone can be 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL or 15 ng / mL, etc., the concentration of the standard solution of 17α-hydroxyprogesterone can be 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL or 15 ng / mL, etc., the concentration of the standard solution of dehydroepiandrosterone sulfate can be 2000 ng / mL, 2100 ng / mL, 2200 ng / mL, 2300 ng / mL, 2400 ng / mL, 2500 ng / mL, 2600 ng / mL, 2700 ng / mL, 2800 ng / mL, 2900 ng / mL or 3000 ng / mL, etc., and the concentration of the standard solution of dehydroepiandrosterone can be 300 ng / mL, 310 ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL or 350 ng / mL, etc., but not limited to the values listed above, and other unlisted values within the above numerical range are equally applicable.

[0024] Preferably, in the high-level quality control reagent, the concentration of the standard solution of testosterone is 600-700 ng / mL, the concentration of the standard solution of androstenedione is 200-300 ng / mL, the concentration of the standard solution of dihydrotestosterone is 1000-1500 ng / mL, the concentration of the standard solution of 17α-hydroxyprogesterone is 1000-1500 ng / mL, the concentration of the standard solution of dehydroepiandrosterone sulfate is 200000-300000 ng / mL, and the concentration of the standard solution of dehydroepiandrosterone is 30000-35000 ng / mL; among them, the concentration of the standard solution of testosterone can be 600 ng / mL, 610 ng / mL, 620 ng / mL, 630 ng / mL, 640 ng / mL, 650 ng / mL, 660 ng / mL, 670 ng / mL, 680 ng / mL, 690 ng / mL or 700 ng / mL, etc., the concentration of the standard solution of androstenedione can be 200 ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 270 ng / mL, 280 ng / mL, 290 ng / mL or 300 ng / mL, etc., the concentration of the standard solution of dihydrotestosterone can be 1000 ng / mL, 1100 ng / mL, 1200 ng / mL, 1300 ng / mL, 1400 ng / mL or 1500 ng / mL, etc., the concentration of the standard solution of 17α-hydroxyprogesterone can be 1000 ng / mL, 1100 ng / mL, 1200 ng / mL, 1300 ng / mL, 1400 ng / mL or 1500 ng / mL, etc., the concentration of the standard solution of dehydroepiandrosterone sulfate can be 200000 ng / mL, 210000 ng / mL, 220000 ng / mL, 230000 ng / mL, 240000 ng / mL, 250000 ng / mL, 260000 ng / mL, 270000 ng / mL, 280000 ng / mL, 290000 ng / mL or 300000 ng / mL, etc., and the concentration of the standard solution of dehydroepiandrosterone can be 30000 ng / mL, 31000 ng / mL, 32000 ng / mL, 33000 ng / mL, 34000 ng / mL or 35000 ng / mL, etc., but not limited to the values listed above, and other unlisted values within the above numerical range are equally applicable.

[0025] Preferably, the internal standard includes testosterone-d3, androstenedione-d7, dihydrotestosterone-d4, 17α-hydroxyprogesterone-d8, dehydroepiandrosterone-d6 and dehydroepiandrosterone sulfate-d6.

[0026] Preferably, the alternative matrix includes any one of low-fat BSA solution, hormone-free serum or hormone-free plasma.

[0027] Preferably, the mass fraction of the low-fat BSA (bovine serum albumin) solution is 3-5%, such as 3%, 3.5%, 4%, 4.5% or 5%, etc., but not limited to the values listed above, and other unlisted values within the above numerical range are equally applicable.

[0028] In a second aspect, the present invention provides the use of the kit as described above in the detection of steroid hormones.

[0029] In a third aspect, the present invention further provides a detection method for simultaneously detecting six steroid hormones, and the detection method is carried out using the kit as described above;

[0030] The detection method includes the following steps:

[0031] (1) Mix the sample to be tested with an internal standard and an extractant, and centrifuge to obtain a first supernatant and a lower supernatant. Mix the lower supernatant with a precipitant, and then centrifuge to obtain a second supernatant. Mix the second supernatant with the residue of the dried first supernatant and a diluent to obtain a test solution;

[0032] (2) Dilute mobile phase additive A and mobile phase additive B respectively to obtain mobile phase A and mobile phase B;

[0033] (3) Perform liquid chromatography-mass spectrometry detection on the test solution and the standard curve solution respectively, and calculate the content of steroid hormones in the sample to be tested according to the detection results in combination with the standard curve;

[0034] Steps (1) and (2) do not distinguish the order;

[0035] The steroid hormones include testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone and 17α-hydroxyprogesterone.

[0036] By adopting a specific kit and combining liquid-liquid extraction and precipitation method, the above detection method realizes the simultaneous extraction and detection of six PCOS markers in the same sample system; compared with the conventional extraction method, this method has low cost, does not require the use of expensive SPE consumables, saves samples; and avoids a large amount of solvent waste. At the same time, the detection range is reasonable and can cover the indicators of most people.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The present invention provides a kit for simultaneously detecting six steroid hormones, which can be used to simultaneously detect testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone and 17α-hydroxyprogesterone, and has the advantages of high accuracy and low quantification limit;

[0039] (2) The present invention also provides a detection method for simultaneously detecting six steroid hormones. By using a specific kit and combining liquid-liquid extraction and precipitation methods, the simultaneous extraction and detection of six PCOS biomarkers in the same sample system are achieved; compared with conventional extraction methods, this method is low-cost, does not require the use of expensive SPE consumables, saves samples; and avoids a large amount of solvent waste. At the same time, the detection range is reasonable and can cover the indicators of most people. Detailed implementation manners

[0040] To further elaborate on the technical means and effects adopted by the present invention, the following further illustrates the technical solution of the present invention in combination with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0041] Example 1

[0042] This example provides a kit and a detection method for simultaneously detecting six steroid hormones. The composition of the kit is as follows:

[0043]

[0044]

[0045] The detection method is as follows:

[0046] Take 200 μL of the sample to be tested and place it in a centrifuge tube. First, add 10 μL of the internal standard solution, then add 1000 μL of the extractant, vortex for 5 min, centrifuge at 14800 rpm for 5 min, and respectively take 800 μL of the supernatant to centrifuge tube A and 50 μL of the lower clear liquid to centrifuge tube B. The supernatant is blown to dryness with liquid nitrogen for standby. Add 200 μL of methanol to the lower clear liquid, vortex for 1 min, centrifuge at 14800 rpm for 5 min, take 50 μL of the supernatant and add it to the centrifuge tube that has been blown dry with nitrogen, and then add 150 μL of the diluent and mix well. Inject 20 μL for analysis.

[0047] Linear and quality control samples:

[0048] Take 10 μL of 20× standard curve solution and quality control reagent, add 190 μL of blank matrix and 10 μL of internal standard solution. Then add 1000 μL of extraction solvent ethyl acetate:n-hexane (1:4, v / v), vortex for 5 min to mix evenly. Centrifuge at 21100×g (14800 rpm) for 5 minutes at 4 °C using a high-speed refrigerated centrifuge. Take 800 μL of the supernatant and blow it to dryness with nitrogen. Add 200 μL of methanol to the lower clear liquid, vortex for 1 min, centrifuge at 21100×g for 5 min. Take 50 μL of the supernatant into the centrifuge tube dried by nitrogen blowing, and then add 150 μL of diluent and mix evenly. Inject 20 μL for analysis.

[0049] The solution concentrations are as follows:

[0050]

[0051] The detection conditions are as follows:

[0052]

[0053]

[0054]

[0055] Using the ratio of the compound peak area to the internal standard peak area as the ordinate and the ratio of the compound concentration to the internal standard concentration as the abscissa, plot the calibration curve. The results of the linear curve are as follows. The linear R values of the six compounds are all greater than 0.99, and the back-calculated values of each point are all within 85 - 115%, meeting the requirements.

[0056]

[0057] The results of measuring two real clinical samples using the above method are as follows:

[0058] 1. Dehydroepiandrosterone sulfate: 2311.20 ng / mL; Dehydroepiandrosterone: 5.91 ng / mL; Androstenedione: 0.69 ng / mL; Testosterone: 0.24 ng / mL; Dihydrotestosterone: 0.12 ng / mL; 17-Hydroxyprogesterone: 0.23 ng / mL;

[0059] 2. Dehydroepiandrosterone sulfate: 2171.98 ng / mL; Dehydroepiandrosterone: 7.55 ng / mL; Androstenedione: 0.71 ng / mL; Testosterone: 0.14 ng / mL; Dihydrotestosterone: 0.09 ng / mL; 17-Hydroxyprogesterone: 0.26 ng / mL.

[0060] Example 2

[0061] This example compared the effects of different pretreatment processes on the detection method, and respectively compared the detection limits, quantification limits, precisions, and recoveries (accuracies) of solid-phase extraction (SPE), liquid-liquid extraction (LLE), protein precipitation (PPT), and post-liquid precipitation (LP, i.e., the method of Example 1). Mixed standard samples with low, medium, and high concentrations were added to human serum samples, and the recoveries of the compounds at low, medium, and high concentration points were respectively compared.

[0062] Solid-phase extraction method: 20 μL of magnetic beads were activated with 1000 μL of methanol, then equilibrated with 1000 μL of water. After adding 200 μL of sample and 10 μL of internal standard, they were successively washed with 500 μL of acetonitrile-water (1:9, v / v) and 500 μL of n-hexane, eluted with 100 μL of methanol-acetonitrile (1:9, v / v), and then 50 μL of water was added and mixed evenly. The injection volume was 20 μL.

[0063] Liquid-liquid extraction method: Take 200 μL of sample, add 10 μL of internal standard and 1000 μL of ethyl acetate-n-hexane (1:4, v / v), vortex for 5 min, centrifuge at 14800 rmp for 5 min, take 800 μL of supernatant, completely dry it with a nitrogen blower, add 100 μL of 60% methanol aqueous solution to dissolve and mix evenly, and then inject it into the machine. The injection volume was 20 μL.

[0064] Protein precipitation method: Take 50 μL of sample and 10 μL of internal standard, add 200 μL of methanol, vortex for 1 min, centrifuge at 14800 rpm for 5 min, take 150 μL of supernatant, add 150 μL of water for dilution, and then inject it into the machine. The injection volume was 20 μL.

[0065] Linearity and accuracy: According to the General Requirements for Kits for the Determination by Liquid Chromatography-Mass Spectrometry, the correlation coefficient (R) of linearity should be not less than 0.9900; the relative deviation of the measurement results of S1-S6, QCL, QCM, and QCH should be within the range of ±15.0%, and the recovery rate should be within the range of 85.0%-115.0%. Through calibration with their respective linear curves, the results are shown in the following table. The SPE, LLE, and PPT methods failed to fully meet the requirements of linear correlation coefficient, accuracy, and recovery rate. Only the linearity and recovery of the LP method met the specified standards.

[0066] Detection limit: Under the comparison of signal-to-noise ratio at the same concentration, the higher the signal-to-noise ratio, the lower the detection limit. Taking the testosterone concentration of 0.01 ng / mL as an example, the signal-to-noise ratios of LP, SPE, LLE, and PPT decrease in turn from high to low; in the sample with the concentration of 0.02 ng / mL androstenedione, the signal-to-noise ratios from high to low are LP, LLE, SPE, and PPT in turn; for the sample with the concentration of 0.5 ng / mL dihydrotestosterone, the signal-to-noise ratios from high to low are LP, LLE, SPE, and PPT in turn; in the sample of 0.1 ng / mL 17-hydroxyprogesterone, the signal-to-noise ratios from high to low are LP, LLE, SPE, and PPT in turn; in the sample of 0.5 ng / mL dehydroepiandrosterone, the signal-to-noise ratios from high to low are LP, LLE, SPE, and PPT in turn; finally, in the sample of 20 ng / mL dehydroepiandrosterone sulfate, the signal-to-noise ratios from high to low are LP, LLE, SPE, and PPT in turn. Through the above comparison, it can be concluded that the LP method has the lowest detection limit.

[0067] Quantitation limit: The quantitation limits of testosterone, androstenedione, and 17-hydroxyprogesterone in SPE, LLE, PPT, and LP methods are the same, which are 0.0125 ng / mL, 0.005 ng / mL, and 0.025 ng / mL respectively. The quantitation limit of dihydrotestosterone under the LP method is 0.025 ng / mL, and the quantitation limits of SPE, LLE, and PPT methods are 0.1 ng / mL; the quantitation limit of dehydroepiandrosterone under SPE and PPT methods is 1 ng / mL, and the quantitation limits of LLE and LP methods are 0.625 ng / mL; the quantitation limits of dehydroepiandrosterone sulfate under SPE and LLE methods are 20 ng / mL, and the quantitation limits of PPT and LP methods are 5 ng / mL.

[0068] Precision: Repeat the samples at 3 concentrations within the linear range 10 times, and the coefficient of variation (CV) should not be greater than 15%. The results are shown in the table. SPE / LLE and PPT methods fail to fully meet the precision requirements, while all six indicators of the LP method can meet the precision requirements.

[0069]

[0070]

[0071]

[0072]

[0073] Example 3

[0074] This example compares the effects of different extractants and extractant ratios during the pretreatment process on the detection. Taking the peak area as the measurement, the results are as follows:

[0075]

[0076]

[0077] Subsequently, mixed standard samples with low, medium, and high concentrations were added to human serum samples, and the recoveries of the compounds at the low, medium, and high concentration points were compared respectively.

[0078]

[0079]

[0080] It can be seen from the above experimental data that different ratios of extractants have little effect on the change in the peak area of DHEA. For the other five compounds, the higher the proportion of ethyl acetate, the better the response. However, as the proportion of ethyl acetate increases, the baseline noise increases, and as the proportion of n-hexane increases, the baseline noise decreases. Considering the response requirements of all substances and combining the recovery data, when the solution ratio is EA:Hex - 1:1 or 1:2, the recoveries of some compounds cannot pass, while when the solvent ratio is 1:4, the recoveries of all compounds can pass.

[0081] The applicant declares that the present invention uses the above embodiments to illustrate the kit for simultaneously detecting six steroid hormones and its application of the present invention. However, the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and the disclosure scope of the present invention.

[0082] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept scope of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0083] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any suitable way. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A kit for simultaneously detecting six steroid hormones, characterized in that, The steroid hormones include testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone, and 17α-hydroxyprogesterone; The kit includes an extraction solution, a mobile phase additive, and a standard quality control product; The extraction solution includes an extractant, a precipitant, and a diluent. The extractant includes ethyl acetate and / or n-hexane. The precipitant includes alcohols and / or acetonitrile. The diluent includes an aqueous solution of alcohols and / or acetonitrile; The mobile phase additive includes mobile phase additive A, and mobile phase additive A is an aqueous ammonium fluoride solution or an ammonium fluoride methanol solution; The standard quality control product includes a standard curve solution, a quality control reagent, an internal standard, and a blank matrix; The standard curve solution includes standard product solutions of testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone, and 17α-hydroxyprogesterone with at least 5 concentration gradients; The quality control reagent includes a low-level quality control reagent and a high-level spiked reagent. The low-level quality control reagent and the high-level quality control reagent are standard product solutions of testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone, and 17α-hydroxyprogesterone; The internal standard is an isotope internal standard of the steroid hormone; The blank matrix is a substitute matrix.

2. The kit according to claim 1, characterized in that, The extractant is ethyl acetate and n-hexane, and the volume ratio of ethyl acetate to n-hexane is 1:(3 - 5); Preferably, the alcohols include methanol and / or ethanol.

3. The kit according to claim 1 or 2, characterized in that, The mobile phase additive further includes mobile phase additive B, and mobile phase additive B is an aqueous ammonium fluoride solution or an ammonium fluoride methanol solution, and mobile phase additive A and mobile phase additive B have different components.

4. The kit according to any one of claims 1 to 3, characterized in that The concentration of the aqueous ammonium fluoride solution is 25 - 250 mM, and the concentration of the ammonium fluoride methanol solution is 25 - 250 mM.

5. The kit according to any one of claims 1-4, characterized in that, In the standard curve solution, the concentration of the standard product solution of testosterone is 0.25 - 1000 ng / mL, the concentration of the standard product solution of androstenedione is 0.1 - 400 ng / mL, the concentration of the standard product solution of dihydrotestosterone is 0.5 - 2000 ng / mL, the concentration of the standard product solution of 17α-hydroxyprogesterone is 0.5 - 2000 ng / mL, the concentration of the standard product solution of dehydroepiandrosterone sulfate is 100 - 400000 ng / mL, and the concentration of the standard product solution of dehydroepiandrosterone is 12.5 - 50000 ng / mL.

6. The kit according to any one of claims 1-5, characterized in that In the low-level quality control reagent, the concentration of the standard product solution of testosterone is 6 - 7 ng / mL, the concentration of the standard product solution of androstenedione is 2 - 3 ng / mL, the concentration of the standard product solution of dihydrotestosterone is 10 - 15 ng / mL, the concentration of the standard product solution of 17α-hydroxyprogesterone is 10 - 15 ng / mL, the concentration of the standard product solution of dehydroepiandrosterone sulfate is 2000 - 3000 ng / mL, and the concentration of the standard product solution of dehydroepiandrosterone is 300 - 350 ng / mL.

7. The kit according to any one of claims 1-6, characterized in that, In the high-level quality control reagent, the concentration of the standard solution of testosterone is 600 - 700 ng / mL, the concentration of the standard solution of androstenedione is 200 - 300 ng / mL, the concentration of the standard solution of dihydrotestosterone is 1000 - 1500 ng / mL, the concentration of the standard solution of 17α-hydroxyprogesterone is 1000 - 1500 ng / mL, the concentration of the standard solution of dehydroepiandrosterone sulfate is 200000 - 300000 ng / mL, and the concentration of the standard solution of dehydroepiandrosterone is 30000 - 35000 ng / mL.

8. The kit according to any one of claims 1-7, characterized in that, The internal standard includes testosterone-d3, androstenedione-d7, dihydrotestosterone-d4, 17α-hydroxyprogesterone-d8, dehydroepiandrosterone-d6, and dehydroepiandrosterone sulfate-d6; Preferably, the alternative matrix includes any one of a low-fat BSA solution, hormone-free serum, or hormone-free plasma; Preferably, the mass fraction of the low-fat BSA solution is 3 - 5%.

9. Use of a kit according to any one of claims 1 - 8 in the detection of steroid hormones.

10. A detection method for simultaneously detecting six steroid hormones, characterized in that, The detection method is carried out using a kit according to any one of claims 1 - 8; The detection method includes the following steps: (1) Mix the sample to be tested with the internal standard and the extractant, centrifuge to obtain a first supernatant and a lower clear liquid. Mix the lower clear liquid with a precipitant, then centrifuge to obtain a second supernatant. Mix the second supernatant with the dried residue of the first supernatant and a diluent to obtain a test solution; (2) Dilute mobile phase additive A and mobile phase additive B respectively to obtain mobile phase A and mobile phase B; (3) Perform liquid chromatography - mass spectrometry detection on the test solution and the standard curve solution respectively, and calculate the content of steroid hormones in the sample to be tested according to the detection results in combination with the standard curve; Steps (1) and (2) do not distinguish the order; The steroid hormones include testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone, and 17α-hydroxyprogesterone.

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