A kit and method for simultaneous detection of 18 steroid hormones based on double derivatization technology

Abstract

The present invention belongs to the field of clinical testing, and specifically relates to a kit and method for simultaneously detecting 18 types of steroid hormones based on a double derivatization technology. An internal standard solution is added to the series of calibration solution, the series of quality control solution and the serum sample respectively; a protein precipitant and a sample extract are then added to each sample, and sample enhancement solution 1 is added to the extract to perform an oximation reaction; sample enhancement solution 2 and a sample conditioning solution are further added to perform an esterification reaction; after the reaction is completed, the reaction solution is centrifuged, and the supernatant is taken, and the supernatant is subjected to LC-MS / MS detection. The present invention adopts a double derivatization method to simultaneously detect multiple steroid hormones. There is no need for positive and negative switching during mass spectrometry detection, and no high-value consumables such as magnetic beads or SPE are required. The simultaneous detection of estrogen and other steroid hormones can be achieved on a conventional liquid chromatography-tandem mass spectrometry platform, which expands the clinical applicability of the steroid hormone detection method.

Description

Technical Field

[0001] The present invention belongs to the field of clinical testing and relates to a clinical detection kit and method for steroid hormones, and specifically to a kit and method for simultaneously detecting 18 types of steroid hormones based on double derivatization technology. Background Art

[0002] Steroid hormones are a class of fat-soluble hormones synthesized from cholesterol, including testosterone, estradiol, and progesterone. They play important regulatory roles in the human body. Abnormal levels of steroid hormones may be associated with abnormalities in other steroid hormones. Therefore, simultaneous testing of multiple steroid hormones can provide a more comprehensive assessment of endocrine function and help identify potential problems. Currently, liquid chromatography-tandem mass spectrometry offers unparalleled advantages over other methods for steroid hormone detection, but it still faces significant challenges.

[0003] A key factor is that some steroid hormones not only have extremely low concentrations in the human body, but also have low ionization efficiency, resulting in poor sensitivity. Currently, only high-end mass spectrometers such as the AB SCIEX 6500 and Waters xevo-TQS can detect steroid hormones at the picogram level, requiring demanding conditions. Another challenge is that aldosterone, along with estradiol, estriol, and other estrogens, not only have extremely low concentrations in the human body, but mass spectrometry detection requires negative ion scanning mode. Therefore, when simultaneously detecting aldosterone, estradiol, estriol, and other steroids, the mass spectrometer requires fast switching between positive and negative modes. Otherwise, sufficient scan points may not be acquired within the chromatographic peak width, affecting quantitative accuracy and placing high demands on mass spectrometer performance. Furthermore, sample processing requires enrichment techniques, which are not only complex but also require expensive solid-phase extraction or magnetic bead consumables, hindering the widespread adoption of steroid hormone testing services in clinical laboratories.

[0004] One solution to addressing the low sensitivity and high cost of steroid hormone detection is to use derivatization to enhance mass spectrometry signal intensity. For example, literature reports that using hydroxylamine and Girard's reagent to derivatize carbonyl-containing steroid hormones can significantly improve mass spectrometry sensitivity. However, these derivatization reagents are incapable of reacting with compounds that do not contain carbonyl groups, such as estradiol and estriol. Literature also reports that dansyl chloride can be used to derivatize the phenolic hydroxyl groups of estradiol and estriol. However, these derivatization reagents are incapable of reacting with the vast majority of steroid hormones other than estrogen, making it impossible to simultaneously detect hormones containing phenolic hydroxyl groups and other hormones using derivatization methods. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the deficiencies of the existing technology and provide a kit for simultaneously detecting 18 steroid hormones based on double derivatization technology.

[0006] Another technical problem to be solved by the present invention is to provide an application of the above-mentioned kit in detecting steroid hormones in serum using high performance liquid chromatography tandem mass spectrometry.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] The present invention provides a kit for simultaneously detecting 18 steroid hormones based on a double derivatization technology, wherein the steroid hormones are 11-deoxycortisol, dehydroepiandrosterone, corticosteroid, 18-hydroxycorticosterone, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, progesterone, cortisol, 21-deoxycortisol, aldosterone, testosterone, androstenedione, corticosterone, estrone, estradiol, estriol and dehydroepiandrosterone sulfate;

[0009] The kit contains the following reagents:

[0010] (1) Series of calibrators:

[0011] The series of calibrators are freeze-dried products of a series of calibrator working solutions with six different concentration levels, and the series of calibrator working solutions are calibrator working solutions with six different concentration levels prepared by diluting a mixed working solution of steroid hormones of known concentration with a second solvent;

[0012] The six concentration levels of the calibrator working solution are:

[0013] The concentrations of pregnenolone, progesterone, estradiol, estriol, 21-deoxycortisol, aldosterone, and testosterone were the same, with the six concentrations being 0.05 ng / mL, 0.125 ng / mL, 0.25 ng / mL, 1.25 ng / mL, 5 ng / mL, and 10 ng / mL;

[0014] The concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosterone, androstenedione, corticosterone, and dehydroepiandrosterone were the same, with the six concentrations being 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 2.5 ng / mL, 10 ng / mL, and 20 ng / mL;

[0015] The concentrations of estrone and 18-hydroxycorticosterone were the same, and the six concentrations were 0.02 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, and 4 ng / mL;

[0016] The six concentrations of dehydroepiandrosterone sulfate were: 50 ng / mL, 125 ng / mL, 250 ng / mL, 1250 ng / mL, 5000 ng / mL, and 10000 ng / mL;

[0017] The six concentrations of cortisol were: 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 25 ng / mL, 100 ng / mL, and 200 ng / mL;

[0018] (2) Series of quality control products:

[0019] The series of quality control products are freeze-dried products of a series of quality control product working solutions with two different concentration levels, and the series of quality control product working solutions are quality control product working solutions with two different concentration levels prepared by diluting a steroid hormone mixed working solution of known concentration with a second solvent;

[0020] The two concentration levels of the quality control working solution are:

[0021] The concentrations of pregnenolone, progesterone, estradiol, estriol, 21-deoxycortisol, aldosterone, and testosterone were the same, with two concentration points of 0.2 ng / mL and 7.5 ng / mL, respectively;

[0022] The concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosterone, androstenedione, corticosterone, and dehydroepiandrosterone were the same, with two concentration points of 0.4 ng / mL and 15 ng / mL, respectively;

[0023] The concentrations of estrone and 18-hydroxycorticosterone were the same, with two concentration points of 0.08 ng / mL and 3 ng / mL, respectively;

[0024] The two concentration points of dehydroepiandrosterone sulfate were 200 ng / mL and 7500 ng / mL;

[0025] The two concentration points of cortisol are: the two concentration points of cortisol are 4 ng / mL and 150 ng / mL;

[0026] (3) Internal standard solution:

[0027] The internal standard solution is a mixture of various steroid hormone standard internal standard stock solutions of known concentrations, and is diluted with a third solvent to form an internal standard solution;

[0028] The concentrations of 11-deoxycortisol-d5, 17-hydroxyprogesterone-d8, corticosterone-d8, androstenedione-d7, testosterone-d3, corticosteroid-d8, estrone-d4, estradiol-d5, estriol-d3, pregnenolone-d4, aldosterone-d7, 18-hydroxycorticosterone-d4, progesterone-d9, 21-deoxycortisol-d8, 17-hydroxypregnenolone-d3, and cortisol-d4 in the internal standard solution are 200 ng / mL, the concentration of dehydroepiandrosterone-d6 is 1 μg / mL, and the concentration of dehydroepiandrosterone sulfate-d6 is 4 μg / mL;

[0029] (4) Sample extraction solution: any one or a combination of n-hexane, methyl tert-butyl ether, and ethyl acetate;

[0030] (5) Protein precipitant: a mixed solvent of acetonitrile and methanol in any volume ratio;

[0031] (6) Sample conditioning solution: a mixed solution of 0.1 mol / L sodium carbonate aqueous solution and 0.1 mol / L sodium bicarbonate aqueous solution with a volume ratio of 1:9 to 9:1;

[0032] (7) Sample enhancement solution 1: hydroxylamine hydrochloride solution, the solvent in the solution is 10% to 20% methanol aqueous solution or 10% to 20% acetonitrile aqueous solution, and the concentration of hydroxylamine hydrochloride in the solution is 0.5 mol / L to 5 mol / L;

[0033] (8) Sample enhancement solution 2: 1,2-dimethylimidazole-5-sulfonyl chloride solution, the solvent in the solution is acetone or ethyl acetate, and the concentration of 1,2-dimethylimidazole-5-sulfonyl chloride in the solution is 0.2 mg / mL to 5 mg / mL;

[0034] (9) Mobile phase A: 0.1vt% formic acid in water;

[0035] (10) Mobile phase B: methanol solution containing 0.1% formic acid or acetonitrile solution containing 0.1% formic acid;

[0036] The calibrator and the quality control product are freeze-dried using the same freeze-drying process, which sequentially includes a pre-freezing stage, sublimation drying, and desorption drying;

[0037] The operating conditions of the pre-freezing stage are shown in Table 1:

[0038] Table 1

[0039]

[0040]

[0041] The operating conditions of the sublimation drying are shown in Table 2:

[0042] Table 2

[0043]

[0044] The operating conditions of the analytical drying are shown in Table 3:

[0045] Table 3

[0046]

[0047] or,

[0048] The calibrator and the quality control product are freeze-dried using the same freeze-drying process, which sequentially includes a pre-freezing stage, sublimation drying, and desorption drying;

[0049] The operating conditions of the pre-freezing stage are shown in Table 4:

[0050] Table 4

[0051]

[0052] The operating conditions of the sublimation drying are shown in Table 5:

[0053] Table 5

[0054]

[0055] The operating conditions of the analytical drying are shown in Table 6:

[0056] Table 6

[0057]

[0058] In some embodiments, the second solvent consists of bovine serum albumin, an antioxidant, a preservative, a buffer and water; the antioxidant is isoascorbic acid; the preservative is citric acid; the buffer is PBS buffer; preferably, the second solvent consists of 1-5% bovine serum albumin, 0.05%-2% isoascorbic acid, 0.05%-2% citric acid, 10% 10×PBS buffer and deionized water; further preferably, the second solvent consists of 1% bovine serum albumin, 0.1% isoascorbic acid, 0.1% citric acid, 10% 10×PBS buffer and deionized water; the third solvent is a mixed solvent of methanol, acetonitrile and isopropanol in any volume ratio; preferably, the third solvent is a mixed solvent of methanol, acetonitrile and isopropanol in a volume ratio of 3:6:1.

[0059] Wherein, when the steroid hormone mixed working solution of known concentration is mixed with the second solvent, the added proportion of the steroid hormone mixed working solution does not exceed 5% of the total volume.

[0060] In some embodiments, the steroid hormone mixed working solution of known concentration is prepared as follows:

[0061] 11-deoxycortisol, dehydroepiandrosterone, cortisol, 18-hydroxycorticosterone, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, progesterone, cortisol, 21-deoxycortisol, aldosterone, testosterone, androstenedione, corticosterone, estrone, estradiol, and estriol were accurately weighed and dissolved in the first solvent to prepare 1 mg / mL standard stock solutions; dehydroepiandrosterone sulfate was accurately weighed and dissolved in the first solvent to prepare 2 mg / mL standard stock solutions;

[0062] The above-mentioned standard stock solutions are then prepared into a steroid hormone mixed working solution using the first solvent; the concentrations of pregnenolone, progesterone, estradiol, estriol, 21-deoxycortisol, aldosterone, and testosterone in the steroid hormone mixed working solution are all 1 μg / mL; the concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosteroid, androstenedione, corticosterone, and dehydroepiandrosterone are all 2 μg / mL; the concentrations of estrone and 18-hydroxycorticosterone are both 400 ng / mL; the concentration of dehydroepiandrosterone sulfate is 1 mg / mL, and the concentration of cortisol is 20 μg / mL.

[0063] In some embodiments, the known concentrations of various steroid hormone standard internal standard stock solutions are prepared as follows:

[0064] 11-deoxycortisol-d5, dehydroepiandrosterone-d6, cortisol-d8, and 18-hydroxycortisol-d4 were accurately weighed and dissolved in the first solvent to prepare 1 mg / mL standard internal standard stock solutions; dehydroepiandrosterone sulfate-d6 was accurately weighed and dissolved in the first solvent to prepare 2 mg / mL standard internal standard stock solutions; pregnenolone-d4, 17-hydroxypregnenolone-d3, 17-hydroxyprogesterone-d8, progesterone-d9, cortisol-d4, 21-deoxycortisol-d8, aldosterone-d7, testosterone-d3, androstenedione-d7, corticosterone-d8 were accurately weighed. 8、 Estrone-d4, estradiol-d5, and estriol-d3 were dissolved in the first solvent and then prepared into 100 μg / mL standard internal standard stock solutions;

[0065] The above-mentioned internal standard stock solutions are mixed and diluted with the third solvent to obtain the internal standard solution.

[0066] In some embodiments, the first solvent is a methanol solution containing 2,6-di-tert-butyl-4-methylphenol; preferably, in the methanol solution containing 2,6-di-tert-butyl-4-methylphenol, the mass volume ratio of 2,6-di-tert-butyl-4-methylphenol to methanol is 1 g:1000 mL.

[0067] In some embodiments, the series of calibrators and the series of quality control working solutions are freeze-dried separately to obtain the series of calibrators and the series of quality control products.

[0068] In some embodiments, preferably, the sample extraction liquid is n-hexane; the protein precipitant is a mixed solvent of acetonitrile and methanol in a volume ratio of 9:1; the sample conditioning liquid is a mixed solution of 0.1 mol / L sodium carbonate aqueous solution and 0.1 mol / L sodium bicarbonate aqueous solution in a volume ratio of 6:4; the sample enhancement liquid 1 is a hydroxylamine hydrochloride solution, the solvent in the solution is 20vt% methanol aqueous solution, and the concentration of hydroxylamine hydrochloride in the solution is 1 mol / L; the sample enhancement liquid 2 is a 1,2-dimethylimidazole-5-sulfonyl chloride solution, the solvent in the solution is acetone, and the concentration of 1,2-dimethylimidazole-5-sulfonyl chloride in the solution is 1 mg / mL; the mobile phase A is a 0.1vt% formic acid aqueous solution; and the mobile phase B is a methanol solution containing 0.1vt% formic acid.

[0069] In some embodiments, preferably, the calibrator and the quality control are freeze-dried using the same freeze-drying process, and the freeze-drying process sequentially includes a pre-freezing stage, sublimation drying, and desorption drying; wherein, the operating conditions of the pre-freezing stage are shown in Table 4; the operating conditions of the sublimation drying are shown in Table 5; and the operating conditions of the desorption drying are shown in Table 6.

[0070] The use of the above-mentioned kit in the simultaneous detection of 18 steroid hormones in serum using high performance liquid chromatography tandem mass spectrometry is also within the scope of protection of the present invention.

[0071] Specifically, the 18 steroid hormones are 11-deoxycortisol, dehydroepiandrosterone, corticosterone, 18-hydroxycorticosterone, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, progesterone, cortisol, 21-deoxycortisol, aldosterone, testosterone, androstenedione, corticosterone, estrone, estradiol, estriol and dehydroepiandrosterone sulfate;

[0072] Before the determination of steroid hormones, the series of calibrators are reconstituted with ultrapure water according to the corresponding six concentration levels of the series of calibrator working solutions to obtain a series of calibrator working solutions; the series of quality control products are reconstituted with ultrapure water according to the corresponding two concentration levels of the series of quality control working solutions to obtain a series of quality control working solutions.

[0073] The kit is stored at -20°C and is taken out from -20°C before use. After returning to room temperature, the components in the kit are taken out.

[0074] Specifically, the steps for detecting 18 steroid hormones in serum include:

[0075] The internal standard solution is added to the series of calibrator solution, the series of quality control solution and the serum sample respectively; then a protein precipitant and a sample extract are added to each sample, centrifuged, and the supernatant is collected; the supernatant is blown dry, and after adding sample enhancement solution 1, an oximation reaction is carried out at 55-65° C.; sample enhancement solution 2 and a sample adjustment solution are continuously added, and an esterification reaction is carried out at 55-65° C.; after the reaction is completed, the reaction solution is centrifuged, the supernatant is collected, and the supernatant is subjected to LC-MS / MS detection.

[0076] Specifically, preferably, the step of detecting 18 steroid hormones in serum includes:

[0077] 20 μL of internal standard solution was added to 0.3 mL of the series of calibrators, 0.3 mL of the series of quality control solutions, and 0.3 mL of serum sample, respectively. 0.3 mL of protein precipitant and 1.2 mL of sample extract were then added to each sample, and the mixture was stirred at 12000 r·min. -1 Centrifuge for 5 minutes and take the supernatant; blow dry the supernatant with nitrogen at 40°C, add 80 μL sample enhancement solution 1 and perform oximation reaction at 60°C for 15 minutes; continue to add 40 μL sample enhancement solution 2 and 30 μL sample adjustment solution and perform esterification reaction at 60°C for 15 minutes; after the reaction is completed, the reaction solution is rotated at 12000 r·min -1 After centrifugation for 3 min, the supernatant was collected and subjected to LC-MS / MS analysis.

[0078] The six concentration levels of the calibrator working solution are:

[0079] The concentrations of pregnenolone, progesterone, estradiol, estriol, 21-deoxycortisol, aldosterone, and testosterone were the same, with the six concentrations being 0.05 ng / mL, 0.125 ng / mL, 0.25 ng / mL, 1.25 ng / mL, 5 ng / mL, and 10 ng / mL;

[0080] The concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosterone, androstenedione, corticosterone, and dehydroepiandrosterone were the same, with the six concentrations being 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 2.5 ng / mL, 10 ng / mL, and 20 ng / mL;

[0081] The concentrations of estrone and 18-hydroxycorticosterone were the same, and the six concentrations were 0.02 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, and 4 ng / mL;

[0082] The six concentrations of dehydroepiandrosterone sulfate were: 50 ng / mL, 125 ng / mL, 250 ng / mL, 1250 ng / mL, 5000 ng / mL, and 10000 ng / mL;

[0083] The six concentrations of cortisol are: 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 25 ng / mL, 100 ng / mL, and 200 ng / mL.

[0084] The two concentration levels of the quality control working solution are:

[0085] The concentrations of pregnenolone, progesterone, estradiol, estriol, 21-deoxycortisol, aldosterone, and testosterone were the same, with two concentration points of 0.2 ng / mL and 7.5 ng / mL, respectively;

[0086] The concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosterone, androstenedione, corticosterone, and dehydroepiandrosterone were the same, with two concentration points of 0.4 ng / mL and 15 ng / mL, respectively;

[0087] The concentrations of estrone and 18-hydroxycorticosterone were the same, with two concentration points of 0.08 ng / mL and 3 ng / mL, respectively;

[0088] The two concentration points of dehydroepiandrosterone sulfate were 200 ng / mL and 7500 ng / mL;

[0089] The two concentration points of cortisol are: 4 ng / mL and 150 ng / mL.

[0090] Wherein, the concentration of 11-deoxycortisol-d5, 17-hydroxyprogesterone-d8, corticosterone-d8, androstenedione-d7, testosterone-d3, corticosteroid-d8, estrone-d4, estradiol-d5, estriol-d3, pregnenolone-d4, aldosterone-d7, 18-hydroxycorticosterone-d4, progesterone-d9, 21-deoxycortisol-d8, 17-hydroxypregnenolone-d3, and cortisol-d4 in the internal standard solution is 200 ng / mL, the concentration of dehydroepiandrosterone-d6 is 1 μg / mL, and the concentration of dehydroepiandrosterone sulfate-d6 is 4 μg / mL.

[0091] The sample extract is any one of n-hexane, methyl tert-butyl ether and ethyl acetate, or a combination thereof, preferably n-hexane.

[0092] The protein precipitant is a mixed solvent of acetonitrile and methanol in any volume ratio, preferably a mixed solvent of acetonitrile and methanol in a volume ratio of 9:1.

[0093] The sample conditioning solution is a mixed solution of 0.1 mol / L sodium carbonate aqueous solution and 0.1 mol / L sodium bicarbonate aqueous solution in a volume ratio of 1:9 to 9:1, preferably a mixed solution of 0.1 mol / L sodium carbonate aqueous solution and 0.1 mol / L sodium bicarbonate aqueous solution in a volume ratio of 6:4.

[0094] Wherein, the sample enhancement solution 1 is a hydroxylamine hydrochloride solution, the solvent in the solution is a 10vt% to 20vt% methanol aqueous solution or a 10vt% to 20vt% acetonitrile aqueous solution, and the concentration of hydroxylamine hydrochloride in the solution is 0.5mol / L to 5mol / L; preferably, the sample enhancement solution 1 is a hydroxylamine hydrochloride solution, the solvent in the solution is a 20vt% methanol aqueous solution, and the concentration of hydroxylamine hydrochloride in the solution is 1mol / L.

[0095] The sample enhancement solution 2 is a 1,2-dimethylimidazole-5-sulfonyl chloride solution, the solvent in the solution is acetone or ethyl acetate, and the concentration of 1,2-dimethylimidazole-5-sulfonyl chloride in the solution is 0.2 mg / mL to 5 mg / mL; preferably, the sample enhancement solution 2 is a 1,2-dimethylimidazole-5-sulfonyl chloride solution, the solvent in the solution is acetone, and the concentration of 1,2-dimethylimidazole-5-sulfonyl chloride in the solution is 1 mg / mL; the mobile phase A is a 0.1vt% formic acid aqueous solution; and the mobile phase B is a methanol solution containing 0.1vt% formic acid.

[0096] Specifically, the chromatographic conditions for the LC-MS / MS detection are as follows:

[0097] Liquid chromatography conditions: Column: Fenome C8 ( 100×2.1mm, 2.6μm), Fenome C18 ( Column temperature: 40°C; mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in methanol or 0.1% formic acid in acetonitrile; gradient elution: 0-0.5 min, 35% mobile phase B; 0.5-5.0 min, 35%-100% mobile phase B; 5.0-5.1 min, 100%-35% mobile phase B; 5.1-7.0 min, 35% mobile phase B; injection volume: 20 μL; acquisition time: 7 min.

[0098] Mass spectrometry conditions: an electrospray ion source was used, the acquisition mode was positive ion mode, the spray voltage was 3500-5500 V, the sheath gas pressure was 35-50 Arb, the auxiliary gas pressure was 6-10 Arb, the evaporation temperature was 450-500°C, the ion transfer tube temperature was 350-400°C, the acquisition mode was multiple reaction monitoring mode, and the acquisition time was 7 min.

[0099] Preferably, the chromatographic conditions for the LC-MS / MS detection are as follows:

[0100] Liquid chromatography conditions: Column: Fenome C8 ( 100×2.1mm, 2.6μm); column temperature: 40°C; mobile phase A: aqueous solution containing 0.1vt% formic acid; mobile phase B: methanol solution containing 0.1vt% formic acid; gradient elution; elution gradient: 0-0.5min, 35% mobile phase B; 0.5-5.0min, 35%-100% mobile phase B; 5.0-5.1min, 100%-35% mobile phase B; 5.1-7.0min, 35% mobile phase B; injection volume: 20μL; acquisition time: 7min.

[0101] The mass spectrometry chromatography conditions are as follows: an electrospray ion source is used, the acquisition mode is positive ion mode, the spray voltage is 3500 V, the sheath gas pressure is 40 Arb, the auxiliary gas pressure is 10 Arb, the evaporation temperature is 450°C, the ion transfer tube temperature is 350°C, the acquisition mode is multiple reaction monitoring mode, and the acquisition time is 7 min.

[0102] Beneficial effects:

[0103] (1) The present invention adopts a double derivatization method to simultaneously detect multiple steroid hormones. No positive or negative switching is required during mass spectrometry detection, and no high-value consumables such as magnetic beads or SPE are required. It can achieve the simultaneous detection of estrogen and other steroid hormones on a conventional liquid chromatography-tandem mass spectrometry platform (such as TSQquantis, SCIEX 4500, etc.), expanding the clinical applicability of steroid hormone detection methods.

[0104] (2) The present invention prepares a steroid hormone detection kit that does not contain consumables such as magnetic beads and SPE, which is used for the detection of various steroid hormones including estrogen, greatly reducing the detection cost.

[0105] (3) In the prior art, the non-derivative method generally requires the use of solid phase extraction cartridges or plates during sample processing. Not only are the consumables expensive, but the operation is also cumbersome. The volume, flow rate, and pressure of the eluent need to be controlled. The non-derivative method has low sensitivity. Some hormones with concentrations at the picogram level require relatively high-end instruments to detect, and the instrument needs to be able to quickly achieve positive and negative switching functions, which limits the development of steroid hormone detection. The derivatization method cannot detect estrogen and other hormones at the same time. The sample can only be divided into two parts and tested for estrogen and other hormones respectively. The kit provided by the present invention does not require SPE and can be directly used for the detection of 18 steroid hormones in human serum. The kit provided by the present invention greatly saves detection costs and improves detection throughput. The kit provided by the present invention does not require high sensitivity of the mass spectrometer for detection. For example, detection can be achieved on AB SCIEX 4500 and TSQ quantis instruments, which reduces detection costs and expands the clinical development of steroid hormones.

[0106] (4) The kit provided by the present invention uses 1,2-dimethylimidazole-5-sulfonyl chloride (DMIS) instead of dansyl chloride in the prior art as the derivatization reagent for estrogen, thereby solving the problems of dansyl chloride ion inhibition and poor specificity. The present invention adopts double derivatization technology to solve the problem that the traditional derivatization method cannot simultaneously detect estrogen and other steroid hormones, and can simultaneously detect androgens, estrogens and progestins at one time without the need for positive and negative switching. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0108] Figure 1 It is a typical derivatization reaction principle of steroid hormones; among them, route a is the oximation reaction principle of carbonyl-containing steroid compounds, and route b is the esterification reaction principle of steroid hormones containing phenolic hydroxyl groups.

[0109] Figure 2 The effect of different concentrations of hydroxylamine hydrochloride solution on the mass spectrometric response of aldosterone; Figure 2 The concentration of hydroxylamine hydrochloride solution in a is 0.5 mol / L; Figure 2 The concentration of hydroxylamine hydrochloride solution in b is 1 mol / L; Figure 2 The concentration of hydroxylamine hydrochloride solution in c is 2 mol / L.

[0110] Figure 3 The effect of different concentrations of 1,2-dimethylimidazole-5-sulfonyl chloride on the mass spectrometric response of estrone, estradiol, and estriol; Figure 3a is the effect of 0.5 mg / mL 1,2-dimethylimidazole-5-sulfonyl chloride solution on the mass spectrometric response of estrone, estradiol, and estriol; Figure 3 b is the effect of 1 mg / mL 1,2-dimethylimidazole-5-sulfonyl chloride solution on the mass spectrometric response of estrone, estradiol, and estriol; Figure 3 c is the effect of 2 mg / mL 1,2-dimethylimidazole-5-sulfonyl chloride solution on the mass spectrometric responses of estrone, estradiol, and estriol.

[0111] Figure 4 The effect of buffer solutions composed of different ratios of sodium carbonate solution and sodium bicarbonate solution on the mass spectrometric responses of estrone, estradiol and estriol; Figure 4 a is the effect of buffer 1 on the mass spectrometric responses of estrone, estradiol, and estriol; Figure 4 b shows the effect of buffer 2 on the mass spectrometric responses of estrone, estradiol, and estriol; Figure 4 c shows the effect of buffer 3 on the mass spectrometric responses of estrone, estradiol and estriol.

[0112] Figure 5 The effects of different chromatographic columns on the mass spectrometric responses of estrone, estradiol and estriol; Figure 5 a is Fei Nuomei C8 ( Effect of the 100×2.1mm, 2.6μm) column on the mass spectrometric responses of estrone, estradiol, and estriol; Figure 5 b is Fei Nuomei C18 ( 100×3mm, 2.6μm) on the mass spectrometric response of estrone, estradiol, and estriol; Figure 5 c shows the effect of Waters BEHC18 (2.1 mm × 50 mm, 1.7 μm) on the mass spectrometric response of estrone, estradiol, and estriol.

[0113] Figure 6 The extracted ion chromatograms of 18 steroid hormones at the limit of quantification concentration are shown in Figure 2.

[0114] Figure 7 The linearity and lower limit of quantification data of 18 steroid hormones are shown in Figure 2. DETAILED DESCRIPTION

[0115] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0116] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0117] In order to achieve these purposes and other advantages according to the present invention, a liquid chromatography-mass spectrometry method based on double derivatization technology is provided for the simultaneous detection of multiple steroid hormones including estrogen, wherein the method comprises the following steps:

[0118] The first solvent used in this embodiment is a methanol solution containing 2,6-di-tert-butyl-4-methylphenol, and the mass volume ratio of 2,6-di-tert-butyl-4-methylphenol to methanol is 1 g:1000 mL.

[0119] The typical derivatization reaction principle of steroid hormones in the embodiment of the present invention is as follows Figure 1 As shown; among them, route a is the oximation reaction principle of carbonyl-containing steroid compounds, and route b is the esterification reaction principle of steroid hormones containing phenolic hydroxyl groups.

[0120] The bovine serum albumin used in the examples of the present invention was purchased from Sigma, catalog number: B2064.

[0121] Example 1:

[0122] 1. Preparation of reagents

[0123] Before establishing LC-MS / MS, steroid hormone standards need to be prepared into stock solutions and mixed working solutions.

[0124] 1.1. Preparation of stock liquid

[0125] (1) Preparation of standard stock solution:

[0126] 11-deoxycortisol, dehydroepiandrosterone, cortisol, 18-hydroxycorticosterone, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, progesterone, cortisol, 21-deoxycortisol, aldosterone, testosterone, androstenedione, corticosterone, estrone, estradiol, and estriol were accurately weighed and dissolved in the first solvent and then diluted to volume to prepare a single standard stock solution of 1 mg / mL. Dehydroepiandrosterone sulfate was accurately weighed and dissolved in the first solvent and then diluted to volume to prepare a single standard stock solution of 2 mg / mL.

[0127] (2) Preparation of standard internal standard stock solution:

[0128] Accurately weigh 11-deoxycortisol-d5, dehydroepiandrosterone-d6, cortisol-d8, and 18-hydroxycorticosterone-d4, each with a specification of 1 mg. After adding 1 mL of the first solvent to dissolve them, they were prepared into 1 mg / mL single standard internal standard stock solutions.

[0129] Accurately weigh 2 mg of dehydroepiandrosterone sulfate-d6 and dissolve it in the first solvent to prepare a 2 mg / mL single standard internal standard stock solution.

[0130] Pregnenolone-d4, 17-hydroxypregnenolone-d3, 17-hydroxyprogesterone-d8, progesterone-d9, cortisol-d4, 21-deoxycortisol-d8, aldosterone-d7, testosterone-d3, androstenedione-d7, corticosterone-d 8、 Estrone-d4, estradiol-d5, and estriol-d3 were respectively added to the first solvent and dissolved to prepare single standard internal standard stock solutions of 100 μg / mL.

[0131] 1.2 Preparation of mixed working solution

[0132] 10 μL of pregnenolone, progesterone, estradiol, estriol, 21-deoxycortisol, aldosterone and testosterone standard stock solutions were transferred; 20 μL of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosterone, androstenedione, corticosterone and dehydroepiandrosterone standard stock solutions were added; 4 μL of estrone and 18-hydroxycorticosterone standard stock solutions, 5 mL of dehydroepiandrosterone sulfate standard stock solution, and 200 μL of cortisol standard stock solution were placed in a 10 mL volumetric flask, and the first solvent was added to the volume to prepare a mixed working solution of 18 steroid hormones, among which pregnenolone, progesterone, The concentrations of estradiol, estriol, 21-deoxycortisol, aldosterone, and testosterone were all 1 μg / mL; the concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosterone, androstenedione, corticosterone, and dehydroepiandrosterone were all 2 μg / mL; the concentrations of estrone and 18-hydroxycorticosterone were both 400 ng / mL; the concentration of dehydroepiandrosterone sulfate was 1 mg / mL, and the concentration of cortisol was 20 μg / mL.

[0133] 1.3 Preparation of mixed internal standard working solution

[0134] 5 μL of 11-deoxycortisol-d5, cortisol-d8 and 18-hydroxycortisol-d4 standard internal standard stock solutions, 25 μL of dehydroepiandrosterone-d6 standard internal standard stock solution and 50 μL of dehydroepiandrosterone sulfate-d6, 17-hydroxyprogesterone-d8, corticosterone-d8, androstenedione-d7, testosterone-d3, estrone-d4, estradiol-d5, estriol-d3, pregnenolone-d4, aldosterone-d7, progesterone-d9, 21-deoxycortisol-d8, 17-hydroxypregnenolone-d3 and cortisol-d4 standard internal standard stock solutions were respectively transferred into a 25 mL volumetric flask and the solution was dissolved in the first solution. The volume was fixed with a 500 mL flask to prepare a mixed internal standard working solution; wherein the concentrations of 11-deoxycortisol-d5, 17-hydroxyprogesterone-d8, corticosterone-d8, androstenedione-d7, testosterone-d3, corticosteroid-d8, estrone-d4, estradiol-d5, estriol-d3, pregnenolone-d4, aldosterone-d7, 18-hydroxycorticosterone-d4, progesterone-d9, 21-deoxycortisol-d8, 17-hydroxypregnenolone-d3, and cortisol-d4 were 200 ng / mL; the concentration of dehydroepiandrosterone-d6 was 1 μg / mL; and the concentration of dehydroepiandrosterone sulfate-d6 was 4 μg / mL.

[0135] 1.4 Preparation of Calibrator Solutions and Quality Controls

[0136] The mixed working solution was diluted 20,000-fold, 8,000-fold, 4,000-fold, 800-fold, 200-fold, and 100-fold with the second solvent to prepare six calibrants. The mixed working solution was diluted 5,000-fold and 133.33-fold with the second solvent to prepare two quality control solutions. The second solvent consisted of bovine serum albumin, isoascorbic acid, citric acid, 10× PBS buffer, and deionized water. The concentrations of bovine serum albumin, isoascorbic acid, and citric acid were 1% (m / v), 0.1% (m / v), and 0.1% (m / v). The volume of 10× PBS in the second solvent was 10%, with the remainder being deionized water. The units of m / v are g / mL.

[0137] The six concentration points of the calibrator solution are: pregnenolone, progesterone, estradiol, estriol, 21-deoxycortisol, aldosterone, and testosterone have the same concentration, and the six concentrations are 0.05 ng / mL, 0.125 ng / mL, 0.25 ng / mL, 1.25 ng / mL, 5 ng / mL, and 10 ng / mL, respectively.

[0138] The two concentration points of the quality control product are: pregnenolone, progesterone, estradiol, estriol, 21-deoxycortisol, aldosterone, and testosterone have the same concentration, and the two concentration points are 0.2 ng / mL and 7.5 ng / mL, respectively.

[0139] The six concentration points of the calibrator solution are: 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosteroid, androstenedione, corticosterone, and dehydroepiandrosterone have the same concentration, and the six concentrations are 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 2.5 ng / mL, 10 ng / mL, and 20 ng / mL, respectively.

[0140] The two concentration points of the quality control product are: the concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosteroid, androstenedione, corticosterone, and dehydroepiandrosterone are the same, and the two concentration points are 0.4 ng / mL and 15 ng / mL respectively.

[0141] The six concentration points of the calibrator solution are: the concentrations of estrone and 18-hydroxycorticosterone are the same, and the six concentrations are 0.02 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, and 4 ng / mL, respectively.

[0142] The two concentration points of the quality control product are: the concentrations of estrone and 18-hydroxycorticosterone are the same, and the two concentration points are 0.08 ng / mL and 3 ng / mL respectively.

[0143] The six concentration points of the calibrator solution are: the six concentrations of dehydroepiandrosterone sulfate are: 50 ng / mL, 125 ng / mL, 250 ng / mL, 1250 ng / mL, 5000 ng / mL, and 10000 ng / mL.

[0144] The two concentration points of the quality control product are: the two concentration points of dehydroepiandrosterone sulfate are 200 ng / mL and 7500 ng / mL respectively.

[0145] The six concentration points of the calibrator solution are: the six concentrations of cortisol are: 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 25 ng / mL, 100 ng / mL, and 200 ng / mL.

[0146] The two concentration points of the quality control product are: the two concentration points of cortisol are 4 ng / mL and 150 ng / mL respectively.

[0147] 2. Optimization of mass spectrometry conditions

[0148] 2.1. Screening of carbonyl derivatization reagents

[0149] There are many commonly used carbon-based derivatization reagents in the prior art, such as hydroxylamine hydrochloride, methoxyamine, Girard's reagent T, and Girard's reagent P. Hydroxylamine hydrochloride, methoxyamine, Girard's reagent T, and Girard's reagent P can all undergo derivatization reactions with carbonyl groups. Among them, the derivatization conditions for methoxyamine are relatively harsh, requiring incubation in a 60°C water bath for 30 minutes (https: / / doi.org / 10.1016 / j.chroma.2019.460709). Girard's reagent T and Girard's reagent P can greatly enhance the mass spectrometric response of steroids after reaction with hydroxylamine. However, excess Girard's reagent is difficult to remove, resulting in significant matrix effects (http: / / dx.doi.org / 10.1016 / j.trac.2012.10.003), requiring SPE to remove interference. Therefore, hydroxylamine hydrochloride is preferably selected as the carbonylation derivatization reagent.

[0150] Pregnenolone (P5), 17-hydroxypregnenolone (17OHP5), 17-hydroxyprogesterone (17α-Hydroxyprogesterone, 17OHP4), progesterone (P4), cortisol (F), 21-deoxycortisol (21-Desoxycortisol, 21DOC), aldosterone (ALD), testosterone (T), androstenedione (A4), corticosterone (B), 11-deoxycortisol (11DOC), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA) sulfate, DHEAS), cortisone (E), 18-hydroxycorticosterone (18OHB), pregnenolone-d4, 17-hydroxypregnenolone-d3, 17-hydroxyprogesterone-d8, progesterone-d9, cortisol-d4, 21-deoxycortisol-d8, aldosterone-d7, testosterone-d3, androstenedione-d7, corticosterone-d8, 11-deoxycortisol-d5, dehydroepiandrosterone-d6, dehydroepiandrosterone sulfate-d6, cortisol-d8, 18-hydroxycorticosterone-d4 undergo derivatization reaction with the derivatization reagent hydroxylamine hydrochloride. The specific operation is as follows:

[0151] (1) Hydroxylamine hydrochloride was mixed with a 20% methanol aqueous solution to prepare a 2 mol / L hydroxylamine hydrochloride solution. 100 μL of the steroid hormone mixed working solution and 100 μL of the mixed internal standard working solution were respectively placed in 1.5 mL centrifuge tubes. 100 μL of the 2 mol / L hydroxylamine hydrochloride solution was added to each centrifuge tube. The mixture was shaken and incubated at 60°C for 15 min for derivatization (to allow the steroid hormone containing a carbonyl functional group to undergo an oximation reaction with hydroxylamine hydrochloride to obtain a derivative of the steroid hormone and hydroxylamine, which was used for optimization of mass spectrometry conditions) to obtain a derivatization reaction solution.

[0152] (2) The derivatization reaction solution obtained in step (1) is detected by high performance liquid chromatography tandem mass spectrometry (LC-MS / MS). The specific chromatographic conditions are as follows:

[0153] HPLC conditions:

[0154] The chromatographic column is Fenome C8, 100mm×2.1mm; mobile phase A is: 0.1vt% formic acid aqueous solution; mobile phase B is: methanol solution containing 0.1vt% formic acid; elution gradient, specific elution parameters are: 0-0.5min, mobile phase B is 10%; 0.5-5.0min, mobile phase B is from 10% to 100%; 5.0-5.1min, mobile phase B is 100% to 35%; 5.1-7.0min, mobile phase B is 10%; injection volume is 20μL; acquisition time is 7min.

[0155] Mass spectrometry conditions:

[0156] An electrospray ion source was used, the acquisition mode was positive ion mode, the spray voltage was 3500-5500 V, the sheath gas pressure was 35-50 Arb; the auxiliary gas pressure was 6-10 Arb; the evaporation temperature was 450-500°C; the ion transfer tube temperature was 350-400°C, and the acquisition mode was multiple reaction monitoring (MRM) mode.

[0157] The mass spectrometry parameters such as MRM ion pairs, transmission voltage (RF Lens), and collision voltage (CE) of the above-mentioned steroid hormone samples and the hydroxylamine derivative hydroxylamine hydrochloride are shown in Table 7. As can be seen from Table 7, 15 steroid hormones and their internal standards, including testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17-hydroxyprogesterone, progesterone, 11-deoxycortisol, corticosterone, 17-hydroxypregnenolone, cortisol, cortisol, pregnenolone, 21-deoxycortisol, aldosterone, and 18-hydroxycorticosterone, can undergo derivatization reaction with hydroxylamine and have ion pair parameters that can be used for quantitative detection.

[0158] Table 7 Mass spectrometric parameters of carbonyl-containing steroid hormones after derivatization with hydroxylamine hydrochloride

[0159]

[0160] 2.2. Screening of phenolic hydroxyl derivatives

[0161] To screen for an optimal derivatization reagent that reacts with steroid hormones containing phenolic hydroxyl groups, estradiol, estriol, estradiol-d5, and estriol-d3 were reacted with derivatization reagents (dansyl chloride, 1,2-dimethylimidazole-5-sulfonyl chloride, or 1,2-dimethylimidazole-4-sulfonyl chloride).

[0162] (1) Preparation of dansyl chloride solution: Mix 5 mg of dansyl chloride with 5 mL of acetone to prepare a dansyl chloride solution with a concentration of 1 mg / mL.

[0163] Preparation of 1,2-dimethylimidazole-5-sulfonyl chloride (DMIS) solution: Mix 5 mg of 1,2-dimethylimidazole-5-sulfonyl chloride with 5 mL of acetone to prepare a 1 mg / mL 1,2-dimethylimidazole-5-sulfonyl chloride solution.

[0164] Preparation of 1,2-dimethylimidazole-4-sulfonyl chloride solution: Mix 5 mg of 1,2-dimethylimidazole-4-sulfonyl chloride with 5 mL of acetone to prepare a 1 mg / mL 1,2-dimethylimidazole-4-sulfonyl chloride solution.

[0165] (2) Take 5 μL of estradiol standard stock solution, 5 μL of estriol standard stock solution, 20 μL of estradiol-d5 standard internal standard stock solution, and 20 μL of estriol-d3 standard internal standard stock solution, respectively, and place them in a 2 mL centrifuge tube. Add 1 mL of methanol to dilute and mix, and then take 100 μL of the mixed liquid and mix with 40 μL of A 1 mg / mL derivatization reagent solution (dansyl chloride solution, 1,2-dimethylimidazole-5-sulfonyl chloride solution or 1,2-dimethylimidazole-4-sulfonyl chloride solution) was mixed, and then 30 μL of 50 mmol / L sodium bicarbonate solution was added, and the pH was adjusted to about 10. The mixture was incubated with shaking at 60°C for 15 min for derivatization (the steroid hormone containing a phenolic hydroxyl functional group undergoes esterification reaction with the dansyl chloride solution or 1,2-dimethylimidazole-5-sulfonyl chloride to obtain a steroid hormone and dansyl chloride or 1,2-dimethylimidazole-5-sulfonyl chloride derivative) to obtain a derivatization reaction solution.

[0166] (3) The derivatization reaction solution obtained in step (2) is detected by high performance liquid chromatography tandem mass spectrometry (LC-MS / MS). The specific chromatographic conditions are as follows:

[0167] HPLC conditions:

[0168] The chromatographic column is Fenome C8, 100mm×2.1mm; mobile phase A is: 0.1vt% formic acid aqueous solution; mobile phase B is: methanol solution containing 0.1vt% formic acid; elution gradient, specific elution parameters are: 0-0.5min, mobile phase B is 10%; 0.5-5.0min, mobile phase B is from 10% to 100%; 5.0-5.1min, mobile phase B is 100% to 35%; 5.1-7.0min, mobile phase B is 10%; injection volume is 20μL; acquisition time is 7min.

[0169] Mass spectrometry conditions:

[0170] An electrospray ion source was used, the acquisition mode was positive ion mode, the spray voltage was 3500-5500 V, the sheath gas pressure was 35-50 Arb; the auxiliary gas pressure was 6-10 Arb; the evaporation temperature was 450-500°C; the ion transfer tube temperature was 350-400°C, and the acquisition mode was multiple reaction monitoring (MRM) mode.

[0171] (4) Experimental results:

[0172] (i) The solutions of estradiol and estriol after reaction with 1,2-dimethylimidazole-4-sulfonyl chloride showed no mass spectrometric response, indicating that steroid hormones containing phenolic hydroxyl functional groups may not undergo derivatization reaction with 1,2-dimethylimidazole-4-sulfonyl chloride.

[0173] (ii) The mass spectrometry parameters such as MRM ion pairs, transmission voltage (RF Lens) and collision voltage (CE) of estradiol, estriol and dansyl chloride derivatives are shown in Table 8.

[0174] Table 8 Mass spectrometric parameters of steroid hormones containing phenolic hydroxyl groups and dansyl chloride derivatives

[0175]

[0176] (iii) The mass spectrometry parameters such as MRM ion pairs, transmission voltage (RFLens) and collision voltage (CE) of estradiol, estriol and 1,2-dimethylimidazole-5-sulfonyl chloride are shown in Table 9.

[0177] Table 9 Mass spectrometric parameters of steroid hormones containing phenolic hydroxyl groups and 1,2-dimethylimidazole-5-sulfonyl chloride derivatives

[0178]

[0179]

[0180] From the data in Tables 8 and 9, it can be seen that the fragment ions of dansyl chloride and estrogen derivatives are both 171, which are poorly characterized. 1,2-dimethylimidazole-5-sulfonyl chloride was selected as the derivatization reagent for steroid hormones containing phenolic hydroxyl groups.

[0181] 2.3 Optimization of estrone-derivative reagents

[0182] Since estrone contains both a carbonyl group and a phenolic hydroxyl group, its properties determine that it can undergo derivatization reactions with hydroxylamine and 1,2-dimethylimidazole-5-sulfonyl chloride at the same time. Therefore, it is necessary to add hydroxylamine hydrochloride and 1,2-dimethylimidazole-5-sulfonyl chloride solutions in sequence for double derivatization.

[0183] (1) Hydroxylamine hydrochloride was mixed with a 20 vol% methanol aqueous solution to prepare a hydroxylamine hydrochloride solution with a concentration of 2 mol / L.

[0184] Preparation of 1,2-dimethylimidazole-5-sulfonyl chloride solution: Mix 1,2-dimethylimidazole-5-sulfonyl chloride with 5 mL of acetone to prepare a 1 mg / mL 1,2-dimethylimidazole-5-sulfonyl chloride solution.

[0185] (2) After diluting the stock solutions of estrone (E1) and estrone-d4, add hydroxylamine hydrochloride solution and incubate. Then add 1,2-dimethylimidazole-5-sulfonyl chloride solution and sodium bicarbonate solution to incubate and establish the LC-MS / MS conditions for estrone.

[0186] 5 μL of estrone (E1) standard stock solution and 20 μL of estrone-d4 internal standard stock solution were placed in a 2 mL centrifuge tube. 1 mL of methanol was added for dilution and mixing. 80 μL of 2 mol / L hydroxylamine hydrochloride solution was added, and the mixture was incubated at 60°C with shaking for 15 minutes. Then, 40 μL of 1 mg / mL 1,2-dimethylimidazole-5-sulfonyl chloride solution and 30 μL of 50 mmol / L sodium bicarbonate solution were added, and the mixture was incubated at 60°C with shaking for 15 minutes to obtain the double derivatization reaction solution.

[0187] (3) The double derivatization reaction solution obtained in step (2) was detected by high performance liquid chromatography tandem mass spectrometry (LC-MS / MS). The specific chromatographic conditions were as follows:

[0188] HPLC conditions:

[0189] The chromatographic column is Fenome C8, 100mm×2.1mm; mobile phase A is: 0.1vt% formic acid aqueous solution; mobile phase B is: methanol solution containing 0.1vt% formic acid; elution gradient, specific elution parameters are: 0-0.5min, mobile phase B is 10%; 0.5-5.0min, mobile phase B is from 10% to 100%; 5.0-5.1min, mobile phase B is 100% to 35%; 5.1-7.0min, mobile phase B is 10%; injection volume is 20μL; acquisition time is 7min.

[0190] Mass spectrometry conditions:

[0191] An electrospray ion source was used, the acquisition mode was positive ion mode, the spray voltage was 3500-5500 V, the sheath gas pressure was 35-50 Arb; the auxiliary gas pressure was 6-10 Arb; the evaporation temperature was 450-500°C; the ion transfer tube temperature was 350-400°C, and the acquisition mode was multiple reaction monitoring (MRM) mode.

[0192] The mass spectrometry parameters such as MRM ion pairs, transmission voltage (RF Lens) and collision voltage (CE) of estrone bis-derivatives and estrone-d4 bis-derivatives are shown in Table 10.

[0193] Table 10 Mass spectrometry parameters of estrone bis-derivatives

[0194] Compound Precursor(m / z) Product(m / z) Dwell Time RF Lens CE estrone 429 365 10 75 25 estrone-d4 433 96 10 75 40

[0195] As can be seen from Table 10, although estrone has hydroxyl and phenolic hydroxyl groups, the derivatization reaction is mainly based on the phenolic hydroxyl group. After estrone (molecular weight 270.37) undergoes derivatization reaction with 1,2-dimethylimidazole-5-sulfonyl chloride (molecular weight 194.6), one molecule of hydrogen chloride (molecular weight 36.5) is removed, and the M+H peak of the derivative is 429.

[0196] 3. Optimization of pre-treatment conditions

[0197] The present invention tests serum samples, processed using liquid-liquid extraction followed by derivatization, with the derivatization step being the key. To achieve the desired results with the dual derivatization technique, optimization of the hydroxylamine derivatization process is crucial, followed by optimization of the derivatization processes for estradiol, estriol, and 1,2-dimethylimidazole-5-sulfonyl chloride. Finally, the order of addition of hydroxylamine and 1,2-dimethylimidazole-5-sulfonyl chloride must be carefully selected.

[0198] 3.1. Optimization of derivatization conditions

[0199] Aldosterone (ALD), which has a lower sensitivity, was selected as the optimization indicator. Hydroxylamine hydrochloride standard was dissolved in 20vt% methanol aqueous solution to prepare 0.5mol / L, 1mol / L, and 2mol / L hydroxylamine hydrochloride solutions, respectively. The specific operation was as follows:

[0200] 1) Select fresh human serum samples, divide them into three portions, 300 μL each, and place them in 2 mL centrifuge tubes;

[0201] 2) 0.3 mL methanol-acetonitrile mixed solution (V 乙腈 :V 甲醇 =9:1) and 1.2 mL of n-hexane, vortexed for 3 min, and centrifuged at 12000 rpm for 5 min for extraction;

[0202] 3) Take the n-hexane extraction layer and place it in a new 2 mL centrifuge tube and blow dry at 40°C under nitrogen;

[0203] 4) Add 80 μL of 0.5 mol / L, 1 mol / L, and 2 mol / L hydroxylamine hydrochloride solutions to each of the three dried centrifuge tubes;

[0204] 5) Incubate the centrifuge tube at 60°C with shaking for 15 minutes;

[0205] 6) Remove the centrifuge tube and transfer the sample to a 96-well plate for LC-MS / MS analysis. The specific LC-MS / MS chromatographic conditions are as follows:

[0206] HPLC conditions:

[0207] The chromatographic column is Fenome C8, 100 mm × 2.1 mm; mobile phase A: 0.1 vol% formic acid in water; mobile phase B: 0.1 vol% formic acid in methanol; elution gradient: 10% mobile phase B from 0 to 0.5 min; 10% to 100% mobile phase B from 0.5 to 5.0 min; 100% to 35% mobile phase B from 5.0 to 5.1 min; 10% mobile phase B from 5.1 to 7.0 min; injection volume: 20 μL; acquisition time: 7 min.

[0208] Mass spectrometry conditions:

[0209] An electrospray ion source was used, the acquisition mode was positive ion mode, the spray voltage was 3500-5500 V, the sheath gas pressure was 35-50 Arb; the auxiliary gas pressure was 6-10 Arb; the evaporation temperature was 450-500°C; the ion transfer tube temperature was 350-400°C, and the acquisition mode was multiple reaction monitoring (MRM) mode.

[0210] The optimization results are as follows Figure 2 As shown in the figure, when 2 mol / L hydroxylamine hydrochloride solution was used, the aldosterone response was the highest, but it was not much different from the response at 1 mol / L concentration, so 1 mol / L hydroxylamine hydrochloride solution was selected as the hydroxyl derivatization reagent.

[0211] 3.2. Optimization of derivatization conditions for 1,2-dimethylimidazole-5-sulfonyl chloride

[0212] 3.2.1. Selection of 1,2-dimethylimidazole-5-sulfonyl chloride concentration

[0213] Estrone, estradiol, and estriol were selected as optimization indicators. 1,2-dimethylimidazole-5-sulfonyl chloride standard was dissolved in acetone to prepare 0.5 mg / mL, 1 mg / mL, and 2 mg / mL 1,2-dimethylimidazole-5-sulfonyl chloride solutions, respectively. The specific operation was as follows:

[0214] 1) Select fresh human serum samples, divide them into three portions, 300 μL each, and place them in 2 mL centrifuge tubes;

[0215] 2) 0.3 mL methanol-acetonitrile mixed solution (V 乙腈 :V 甲醇 =9:1) and 1.2 mL of n-hexane, vortexed for 3 min, and centrifuged at 12000 rpm for 5 min for extraction;

[0216] 3) Take the n-hexane extraction layer and place it in a new 2 mL centrifuge tube and blow dry at 40°C under nitrogen;

[0217] 4) Add 80 μL of 0.5 mg / mL, 1 mg / mL, and 2 mg / mL 1,2-dimethylimidazole-5-sulfonyl chloride solution and 60 μL of 0.1 mol / L sodium bicarbonate solution to each of the three dried centrifuge tubes;

[0218] 5) Incubate the centrifuge tube at 60°C with shaking for 15 minutes;

[0219] 6) Remove the centrifuge tube and transfer the sample to a 96-well plate for LC-MS / MS analysis. The specific LC-MS / MS chromatographic conditions are as follows:

[0220] HPLC conditions:

[0221] The chromatographic column is Fenome C8, 100 mm × 2.1 mm; mobile phase A: 0.1 vol% formic acid in water; mobile phase B: 0.1 vol% formic acid in methanol; elution gradient: 10% mobile phase B from 0 to 0.5 min; 10% to 100% mobile phase B from 0.5 to 5.0 min; 100% to 35% mobile phase B from 5.0 to 5.1 min; 10% mobile phase B from 5.1 to 7.0 min; injection volume: 20 μL; acquisition time: 7 min.

[0222] Mass spectrometry conditions:

[0223] An electrospray ion source was used, the acquisition mode was positive ion mode, the spray voltage was 3500-5500 V, the sheath gas pressure was 35-50 Arb; the auxiliary gas pressure was 6-10 Arb; the evaporation temperature was 450-500°C; the ion transfer tube temperature was 350-400°C, and the acquisition mode was multiple reaction monitoring (MRM) mode.

[0224] The optimization results are as follows Figure 3 As shown in the figure, when using different 2 mg / mL concentrations of 1,2-dimethylimidazole-5-sulfonyl chloride solutions, the responses of estrone, estradiol, and estriol were the highest, but the difference with the response of 1 mg / mL concentration was not much, so the 1 mg / mL concentration of 1,2-dimethylimidazole-5-sulfonyl chloride solution was selected as the derivatization reagent for estrone, estradiol, and estriol. Note: Figure 3 Among them, E1 is estrone, E2 is estradiol, E3 is estriol, and DMIS is 1,2-dimethylimidazole-5-sulfonyl chloride.

[0225] 3.2.2 Optimization of the ratio of sodium carbonate solution to sodium bicarbonate solution in the buffer solution

[0226] Because the derivatization reaction of estrone, estradiol, and estriol with 1,2-dimethylimidazole-5-sulfonyl chloride requires an alkaline environment, sodium carbonate, sodium bicarbonate, or a mixture of the two is generally used to provide this alkaline environment. Long-term storage of sodium carbonate will slowly react with carbon dioxide in the air to form sodium bicarbonate, so this protocol uses a mixture of sodium carbonate and sodium bicarbonate as the buffer for the derivatization reaction.

[0227] 1) Preparation of sodium carbonate and sodium bicarbonate solutions

[0228] Weigh 0.53 g of sodium carbonate, place it in a 50 mL centrifuge tube, and add 50 mL of ultrapure water to make a sodium carbonate solution with a concentration of 0.1 mol / L. Weigh 0.42 g of sodium bicarbonate, place it in a 50 mL centrifuge tube, and add 50 mL of ultrapure water to make a sodium bicarbonate solution with a concentration of 0.1 mol / L.

[0229] 2) Preparation of buffer solution.

[0230] 0.1 mol / L sodium carbonate solution and 0.1 mol / L sodium bicarbonate solution were mixed in different volumes to prepare buffer solutions.

[0231] Buffer 1: 0.1 mol / L sodium carbonate solution and 0.1 mol / L sodium bicarbonate solution in a volume ratio of 8:2;

[0232] Buffer 2: 0.1 mol / L sodium carbonate solution and 0.1 mol / L sodium bicarbonate solution in a volume ratio of 7:3;

[0233] Buffer solution 3: The volume ratio of 0.1 mol / L sodium carbonate solution to 0.1 mol / L sodium bicarbonate solution is 6:4.

[0234] 3) The specific operations of the derivation process are as follows:

[0235] a. Select fresh human serum samples and divide them into 3 portions, 300 μL each, and place them in 2 mL centrifuge tubes; 0.3 mL of methanol-acetonitrile mixed solution (V 乙腈 :V 甲醇 =9:1) and 1.2 mL of n-hexane, vortexed for 3 min, and centrifuged at 12000 rpm for 5 min for extraction;

[0236] b. Take the n-hexane extract layer and place it in a new 2 mL centrifuge tube and blow dry at 40°C under nitrogen;

[0237] c. Add 80 μL of 1 mg / mL 1,2-dimethylimidazole-5-sulfonyl chloride solution (solvent: acetone) to each of the three dried centrifuge tubes.

[0238] d. Add 60 μL of buffer 1, buffer 2, and buffer 3 to each centrifuge tube;

[0239] e. Place the centrifuge tube in a shaking incubator at 60°C for 15 minutes;

[0240] f. Remove the centrifuge tube and transfer the sample to a 96-well plate for LC-MS / MS analysis. The specific LC-MS / MS chromatographic conditions are as follows:

[0241] HPLC conditions:

[0242] The chromatographic column is Fenome C8, 100 mm × 2.1 mm; mobile phase A: 0.1 vol% formic acid in water; mobile phase B: 0.1 vol% formic acid in methanol; elution gradient: 10% mobile phase B from 0 to 0.5 min; 10% to 100% mobile phase B from 0.5 to 5.0 min; 100% to 35% mobile phase B from 5.0 to 5.1 min; 10% mobile phase B from 5.1 to 7.0 min; injection volume: 20 μL; acquisition time: 7 min.

[0243] Mass spectrometry conditions:

[0244] An electrospray ion source was used, the acquisition mode was positive ion mode, the spray voltage was 3500-5500 V, the sheath gas pressure was 35-50 Arb; the auxiliary gas pressure was 6-10 Arb; the evaporation temperature was 450-500°C; the ion transfer tube temperature was 350-400°C, and the acquisition mode was multiple reaction monitoring (MRM) mode.

[0245] The optimization results are as follows Figure 4 As shown in the figure, among the different buffer components, when the ratio of sodium carbonate to sodium bicarbonate is 6:4, estrone, estradiol and estriol respond best, so the ratio of sodium carbonate solution to sodium bicarbonate solution in the buffer solution is selected to be 6:4. Figure 4 Among them, E1 is estrone, E2 is estradiol, E3 is estriol, and DMIS is 1,2-dimethylimidazole-5-sulfonyl chloride.

[0246] 3.3. Optimization of the order of derivatization agents

[0247] When carbonyl-containing steroid hormones and phenolic hydroxyl-containing steroid hormones are treated simultaneously, hydroxylamine hydrochloride may affect the derivatization process of estradiol and estriol. Similarly, 1,2-dimethylimidazole-5-sulfonyl chloride solution may inhibit the derivatization reaction of other steroids. Therefore, it is necessary to consider the order of adding hydroxylamine hydrochloride and 1,2-dimethylimidazole-5-sulfonyl chloride to ensure that all steroid hormones can undergo specific derivatization reactions.

[0248] (1) The derivatization process is as follows:

[0249] a) Take 0.3 mL of sample (the lowest concentration of the calibrator) and place it in a 2 mL centrifuge tube. Add 0.3 mL of methanol-acetonitrile mixed solution (V 乙腈 :V 甲醇 =9:1) and 1.2 mL of n-hexane, vortexed for 3 min, and centrifuged at 12000 rpm for 5 min for extraction;

[0250] b) Place the entire n-hexane extract into a new 2 mL centrifuge tube and blow dry at 40°C under nitrogen.

[0251] c) Add 80 μL of 1 mol / L hydroxylamine hydrochloride solution (solvent is 20 vol% methanol in water) to the dry centrifuge tube and incubate with shaking at 60° C. for 15 min;

[0252] d) Add 40 μL of 1 mg / mL 1,2-dimethylimidazole-5-sulfonyl chloride solution and 30 μL of buffer (0.1 mol / L sodium carbonate solution: 0.1 mol / L sodium bicarbonate solution, volume ratio 6:4), incubate at 60°C with shaking for 15 min, and remove the incubated sample.

[0253] e) The incubated sample was centrifuged at 12,000 rpm for 3 minutes, and 100 μL of the supernatant was collected for LC-MS / MS analysis. The specific LC-MS / MS chromatographic conditions were as follows:

[0254] LC-MS / MS detection and analysis, the specific chromatographic conditions are as follows:

[0255] HPLC conditions:

[0256] The chromatographic column is Fenome C8, 100 mm × 2.1 mm; mobile phase A: 0.1 vol% formic acid in water; mobile phase B: 0.1 vol% formic acid in methanol; elution gradient: 10% mobile phase B from 0 to 0.5 min; 10% to 100% mobile phase B from 0.5 to 5.0 min; 100% to 35% mobile phase B from 5.0 to 5.1 min; 10% mobile phase B from 5.1 to 7.0 min; injection volume: 20 μL; acquisition time: 7 min.

[0257] Mass spectrometry conditions:

[0258] An electrospray ion source was used, the acquisition mode was positive ion mode, the spray voltage was 3500-5500 V, the sheath gas pressure was 35-50 Arb; the auxiliary gas pressure was 6-10 Arb; the evaporation temperature was 450-500°C; the ion transfer tube temperature was 350-400°C, and the acquisition mode was multiple reaction monitoring (MRM) mode.

[0259] (2) In order to better optimize the impact of each step of the derivatization reaction on the entire sample processing process, five sample processing methods were designed during the derivatization process:

[0260] Pretreatment method 1: After the sample was extracted with n-hexane and dried, 80 μL of hydroxylamine hydrochloride solution was added, and the sample was incubated with shaking at 60° C. for 15 minutes, and then the sample was injected for analysis.

[0261] Pretreatment 2 method: After the sample was extracted with n-hexane and dried, 40 μL of 1,2-dimethylimidazole-5-sulfonyl chloride solution and 30 μL of buffer were added, incubated with shaking at 60°C for 15 minutes, and then sampled and analyzed.

[0262] Pretreatment 3: After the sample was extracted with n-hexane and dried, 40 μL of 1,2-dimethylimidazole-5-sulfonyl chloride solution and 30 μL of buffer were added, and the mixture was incubated at 60°C with shaking for 15 min. Then, 80 μL of hydroxylamine hydrochloride solution was added, and the mixture was incubated at 60°C with shaking for 15 min before injection for analysis.

[0263] Treatment 4: After the sample was extracted with n-hexane and dried, 80 μL of hydroxylamine hydrochloride solution was added and incubated at 60°C for 15 min. Then, 40 μL of 1,2-dimethylimidazole-5-sulfonyl chloride solution and 30 μL of buffer were added and incubated at 60°C for 15 min. The sample was then injected for analysis.

[0264] Pretreatment method 5: After the sample was extracted with n-hexane and dried, 80 μL of hydroxylamine hydrochloride solution, 40 μL of 1,2-dimethylimidazole-5-sulfonyl chloride solution and 30 μL of buffer solution were added simultaneously, incubated with shaking at 60°C for 15 minutes, and then sampled and analyzed.

[0265] After the samples (calibrator C1, i.e., the sample with the lowest calibrator concentration) were treated with the five pretreatment methods, the peak areas of 18 steroid hormones were collected. The results are shown in Table 11.

[0266] Table 11 Responses (peak areas) of 18 steroid hormones treated with five sample treatment methods

[0267]

[0268]

[0269] The experimental results are shown in Table 11. It can be seen from the table that in the method of pretreatment 1, estrogen (estrone, estradiol, estriol) was not detected; in the method of pretreatment 2, testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17-hydroxyprogesterone, progesterone, 11-deoxycortisol, corticosterone, 17-hydroxypregnenolone, corticosterone, cortisol, pregnenolone, 21-deoxycortisol, aldosterone, and 18-hydroxycorticosterone were not detected; in the methods of pretreatment 3, pretreatment 4, and pretreatment 5, all 18 steroid hormones were detected.

[0270] Among them, the responses of corticosteroids, aldosterone, and 17-hydroxypregnenolone in the pretreatment 3 method were low, significantly lower than those in the pretreatment 1 method. The possible reason was that the pH value in the derivatization system was too high when the steroid hormones reacted with hydroxylamine, which inhibited the derivatization reaction.

[0271] Among them, the response of estrone in pretreatment 4 was significantly lower than that in pretreatment 2 and pretreatment 3. The possible reason was that the response was low due to the influence of hydroxylamine during the derivatization reaction between estrone and 1,2-dimethylimidazole-5-sulfonyl chloride.

[0272] Among them, the responses of almost all steroid hormones in the pre-treatment method 5 were lower than those in the pre-treatment method 4. The possible reason was that the addition of two derivatization reagents at the same time destroyed the specificity of the reaction and led to the inhibition of the derivatization reaction.

[0273] In summary, the pre-treatment method 4 was selected as the pre-treatment method for the samples of the present invention.

[0274] 4. Optimization of LC-MS conditions

[0275] After determining the pretreatment conditions in the above section "3. Optimization of Pretreatment Conditions", it is necessary to optimize the liquid phase conditions of the 18 steroids, including the chromatographic column, mobile phase, and elution gradient, to achieve optimal chromatographic peak responses for all compounds and internal standards.

[0276] Since the concentrations of estrogens (estrone, estradiol, and estriol) are relatively low, estrone, estradiol, and estriol (all at a concentration of 1 ng / mL, with methanol as the solvent) were selected as column optimization indicators, and the column with the best response was chosen.

[0277] In this experiment, 50 μL of different estrogen methanol solutions (estrone, estradiol, and estriol at a concentration of 1 ng / mL, in methanol) were used as samples, 1 mol / L hydroxylamine hydrochloride solution was used as the hydroxyl derivatization reagent, 1 mg / mL 1,2-dimethylimidazole-5-sulfonyl chloride solution was used as the estrogen, estradiol, and estriol derivatization reagent, and a mixture of 0.1 mol / L sodium carbonate solution and 0.1 mol / L sodium bicarbonate solution in a volume ratio of 6:4 was used as the buffer. Sample treatment method 4 was used for sample treatment (treatment method 4: after the sample was extracted with n-hexane and blown dry, 80 μL of hydroxylamine hydrochloride solution was first added, and the mixture was incubated with shaking at 60°C for 15 minutes. Then, 40 μL of 1,2-dimethylimidazole-5-sulfonyl chloride solution and 30 μL of buffer were added, and the mixture was incubated with shaking at 60°C for 15 minutes. Sample injection was then performed). LC-MS / MS detection and analysis were performed using the chromatographic conditions described in "3. Optimization of Pretreatment Conditions."

[0278] (1) When other chromatographic conditions remain unchanged, the chromatographic column is selected as Fenome C8 ( 100×2.1mm, 2.6μm) chromatographic column, Fei Nuo Mei C18 ( 100×3 mm, 2.6 μm) and Waters BEH C18 (2.1 mm×50 mm, 1.7 μm).

[0279] The optimization results are as follows Figure 5 As shown, using Fenome C8 ( 100×2.1mm, 2.6μm) column, the responses of estrone, estradiol and estriol were all high, so Fei Nuomei C8 ( A 100×2.1mm, 2.6μm column was selected as the optimal column. Note: In the figure, Kintex C18 refers to Fenome C18, Kintex C8 refers to Fenome C8, and Waters BEH C18 refers to Waters BEH C18. In the figure, E1 refers to estrone, E2 refers to estradiol, E3 refers to estriol, and DMIS refers to 1,2-dimethylimidazole-5-sulfonyl chloride.

[0280] (2) With other chromatographic conditions remaining unchanged, mobile phase A is an aqueous solution containing 0.1 vt% formic acid, and mobile phase B is a methanol solution containing 0.1 vt% formic acid or an acetonitrile solution containing 0.1 vt% formic acid.

[0281] Both methanol and acetonitrile can be used as mobile phases for steroid hormones. Since methanol is relatively cheap, in a more preferred scheme, mobile phase A is selected to be an aqueous solution containing 0.1vt% formic acid, and mobile phase B is selected to be a methanol solution containing 0.1vt% formic acid.

[0282] (3) Although testosterone and isomers such as dehydroepiandrosterone, 11-deoxycortisol, corticosterone, and 21-deoxycortisol form different characteristic fragment ions after derivatization with hydroxylamine, optimizing the liquid phase gradient to achieve baseline analysis of isomers can enhance the specificity of the method.

[0283] Preferred solution 1: acquisition time 7 minutes; elution gradient: 0-0.5 minutes, mobile phase B is 10%; 0.5-5.0 minutes, mobile phase B from 10% to 100%; 5.0-5.1 minutes, mobile phase B is 100% to 10%; 5.1-7.0 minutes, mobile phase B is 10%; injection volume is 20 μL.

[0284] Preferred solution 2: acquisition time 6 min; elution gradient: 0-0.5 min, mobile phase B is 35%; 0.5-5.0 min, mobile phase B from 35% to 100%; 5.0-5.1 min, mobile phase B is 100% to 35%; 5.1-6.0 min, mobile phase B is 35%; injection volume is 20 μL.

[0285] Preferred solution three: acquisition time 7 min; elution gradient: 0-0.5 min, mobile phase B is 35%; 0.5-5.0 min, mobile phase B from 35% to 100%; 5.0-5.1 min, mobile phase B is 100% to 35%; 5.1-7.0 min, mobile phase B is 35%; injection volume is 20 μL.

[0286] Preferred solution 4: acquisition time 7 min; elution gradient: 0-0.5 min, mobile phase B is 50%; 0.5-5.0 min, mobile phase B from 50% to 100%; 5.0-5.1 min, mobile phase B is 100% to 50%; 5.1-7.0 min, mobile phase B is 50%; injection volume is 20 μL.

[0287] In preferred scheme 1, the separation degree of testosterone and dehydroepiandrosterone, 11-deoxycortisol, corticosterone, and 21-deoxycortisol is high, but the retention times of 17-hydroxyprogesterone and testosterone, progesterone, and androstenedione are all greater than 6.5 minutes. This scheme requires at least 8 minutes of detection time. The possible reason is that the initial mobile phase ratio is too low, resulting in excessive retention time of the compounds.

[0288] In the preferred scheme 2, the separation degree of testosterone and dehydroepiandrosterone, 11-deoxycortisol, corticosterone, and 21-deoxycortisol is worse than that of the scheme 1, but baseline separation can be achieved. The retention time of all compounds is ≤6 minutes, among which the retention time of progesterone is 6 minutes, and the equilibrium time is too short.

[0289] In the fourth preferred embodiment, baseline separation of testosterone, dehydroepiandrosterone, 11-deoxycortisol, corticosterone, and 21-deoxycortisol cannot be achieved.

[0290] Therefore, the initial proportion of mobile phase B was set to 35% and the final equilibrium time of the gradient was changed to 2 min, which resulted in the preferred solution 3. Therefore, the preferred solution 3 was selected as the liquid phase condition.

[0291] (4) The column temperature of this method has little effect on the liquid phase conditions, and the column temperature of the chromatographic column is selected to be 40℃.

[0292] (5) The present invention utilizes double derivatization technology to derivatize all steroid hormones containing carbonyl and phenolic hydroxyl groups during sample processing. Therefore, the ion source in the present invention only needs to use the positive ion mode.

[0293] In a preferred embodiment, an electrospray ion source is used, the collection mode is positive ion mode, the spray voltage is 3500-5500 V, the sheath gas pressure is 35-50 Arb; the auxiliary gas pressure is 6-10 Arb; the evaporation temperature is 450-500°C; the ion transfer tube temperature is 350-400°C, the collection mode is multiple reaction monitoring mode (MRM), and the collection time is 7 minutes.

[0294] In a more preferred embodiment, an electrospray ion source is used, the acquisition mode is positive ion mode, the spray voltage is 3500 V, the sheath gas pressure is 40 Arb; the auxiliary gas pressure is 10 Arb; the evaporation temperature is 450°C; the ion transfer tube temperature is 350°C, the acquisition mode is multiple reaction monitoring mode (MRM), and the acquisition time is 7 minutes.

[0295] 5. Preparation of the kit

[0296] The kit components of a steroid hormone include calibrators of 6 concentrations, quality control products of 2 concentrations, internal standard solution, sample extraction solution, protein precipitation agent, sample adjustment solution, sample enhancement solution 1, sample enhancement solution 2, mobile phase A, and mobile phase B.

[0297] 5.1 Preparation of Calibrators and Quality Controls

[0298] (1) Preparation of standard stock solutions: Dissolve the powders of 18 steroid hormone standards in the first solvent to prepare individual stock solutions. The specific preparation process is as follows:

[0299] 11-deoxycortisol, dehydroepiandrosterone, cortisol, 18-hydroxycorticosterone, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, progesterone, cortisol, 21-deoxycortisol, aldosterone, testosterone, androstenedione, corticosterone, estrone, estradiol, and estriol were accurately weighed and dissolved in the first solvent and then diluted to volume to prepare a single standard stock solution of 1 mg / mL. Dehydroepiandrosterone sulfate was accurately weighed and dissolved in the first solvent and then diluted to volume to prepare a single standard stock solution of 2 mg / mL.

[0300] The first solvent used is a methanol solution containing 2,6-di-tert-butyl-4-methylphenol, and the mass volume ratio of 2,6-di-tert-butyl-4-methylphenol to methanol is 1 g:1000 mL.

[0301] (2) Bovine serum albumin, antioxidants, preservatives, etc. are mixed to form a second solvent. Preferably, the second solvent consists of bovine serum albumin, isoascorbic acid, citric acid, 10×PBS buffer, and deionized water; wherein the concentration of bovine serum albumin is 1% (m / v), the concentration of isoascorbic acid is 0.1% (m / v), the concentration of citric acid is 0.1% (m / v), the volume proportion of 10×PBS in the second solvent is 10%, and the balance is deionized water. The unit of m / v is g / mL.

[0302] (3) The single standard stock solutions of 18 steroid hormones are mixed and then added to the first solvent to prepare a steroid hormone mixed working solution.

[0303] The mixed working solution is prepared as follows:

[0304] 10 μL of pregnenolone, progesterone, estradiol, estriol, 21-deoxycortisol, aldosterone and testosterone standard stock solutions were transferred; 20 μL of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosterone, androstenedione, corticosterone and dehydroepiandrosterone standard stock solutions were added; 4 μL of estrone and 18-hydroxycorticosterone standard stock solutions, 5 mL of dehydroepiandrosterone sulfate standard stock solution, and 200 μL of cortisol standard stock solution were placed in a 10 mL volumetric flask, and the first solvent was added to the volume to prepare a mixed working solution of 18 steroid hormones, among which pregnenolone, progesterone, The concentrations of estradiol, estriol, 21-deoxycortisol, aldosterone, and testosterone were all 1 μg / mL; the concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosterone, androstenedione, corticosterone, and dehydroepiandrosterone were all 2 μg / mL; the concentrations of estrone and 18-hydroxycorticosterone were both 400 ng / mL; the concentration of dehydroepiandrosterone sulfate was 1 mg / mL, and the concentration of cortisol was 20 μg / mL.

[0305] (4) Mix different volumes of the mixed working solution with the second solvent to prepare calibrators C1, C2, C3, C4, C5, C6, and quality control products QC1 and QC2. When the mixed working solution is mixed with the second solvent, the proportion of the mixed working solution added does not exceed 5% of the total volume.

[0306] The preparation method is shown in Table 12:

[0307] Table 12

[0308]

[0309] Among them, in standard samples C1 to C6, the concentrations of pregnenolone, progesterone, estradiol, estriol, 21-deoxycortisol, aldosterone, and testosterone are the same, and the six concentrations are 0.05 ng / mL, 0.125 ng / mL, 0.25 ng / mL, 1.25 ng / mL, 5 ng / mL, and 10 ng / mL, respectively; the concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosterone, androstenedione, corticosterone, and dehydroepiandrosterone are the same, and the six concentrations are 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 2.5 ng / mL, 10 ng / mL, and 2 0ng / mL; the concentrations of estrone and 18-hydroxycorticosterone were the same, and the six concentrations were 0.02ng / mL, 0.05ng / mL, 0.1ng / mL, 0.5ng / mL, 2ng / mL, and 4ng / mL; the six concentrations of dehydroepiandrosterone sulfate were 50ng / mL, 125ng / mL, 250ng / mL, 1250ng / mL, 5000ng / mL, and 10000ng / mL; the six concentrations of cortisol were 1ng / mL, 2.5ng / mL, 5ng / mL, 25ng / mL, 100ng / mL, and 200ng / mL.

[0310] In quality control products QC1 and QC2, the concentrations of pregnenolone, progesterone, estradiol, estriol, 21-deoxycortisol, aldosterone, and testosterone are the same, with two concentration points of 0.2 ng / mL and 7.5 ng / mL, respectively; the concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosterone, androstenedione, corticosterone, and dehydroepiandrosterone are the same, with two concentration points of 0.4 ng / mL and 15 ng / mL, respectively; the concentrations of estrone and 18-hydroxycorticosterone are the same, with two concentration points of 0.08 ng / mL and 3 ng / mL, respectively; the two concentration points of dehydroepiandrosterone sulfate are 200 ng / mL and 7500 ng / mL, respectively; and the two concentration points of cortisol are 4 ng / mL and 150 ng / mL, respectively.

[0311] After the preparation of calibrators and quality control products, each 0.5 mL was dispensed into brown penicillin bottles and freeze-dried.

[0312] 5.2 Preparation of internal standard solution

[0313] (1) 18 steroid hormone internal standard substances are dissolved in a first solvent to prepare individual standard internal standard stock solutions.

[0314] Among them, the 18 steroid hormone internal standard substances are pregnenolone-d4, 17-hydroxypregnenolone-d3, 17-hydroxyprogesterone-d8, progesterone-d9, estradiol-d5, estriol-d3, cortisol-d8, 21-deoxycortisol-d8, aldosterone-d7, testosterone-d3, androstenedione-d7, estrone-d4, corticosterone-d8, 11-deoxycortisol-d5, dehydroepiandrosterone-d5, dehydroepiandrosterone sulfate-d6, corticosterone-d8, and 18-hydroxycorticosterone-d4. The specific preparation process is as follows:

[0315] Accurately weigh 11-deoxycortisol-d5, dehydroepiandrosterone-d6, cortisol-d8, and 18-hydroxycorticosterone-d4, each with a specification of 1 mg. After adding 1 mL of the first solvent to dissolve them, they were prepared into 1 mg / mL single standard internal standard stock solutions.

[0316] Accurately weigh 2 mg of dehydroepiandrosterone sulfate-d6 and dissolve it in the first solvent to prepare a 2 mg / mL single standard internal standard stock solution.

[0317] Pregnenolone-d4, 17-hydroxypregnenolone-d3, 17-hydroxyprogesterone-d8, progesterone-d9, cortisol-d4, 21-deoxycortisol-d8, aldosterone-d7, testosterone-d3, androstenedione-d7, corticosterone-d 8、 Estrone-d4, estradiol-d5, and estriol-d3 were respectively added to the first solvent and dissolved to prepare single standard internal standard stock solutions of 100 μg / mL.

[0318] The first solvent is a methanol solution containing 2,6-di-tert-butyl-4-methylphenol, and the mass volume ratio of 2,6-di-tert-butyl-4-methylphenol to methanol is 1 g:1000 mL.

[0319] (2) Preparation of internal standard solution: 18 steroid hormone standard internal standard stock solutions were mixed and diluted with a third solvent to form an internal standard solution. The third solvent was a mixed solvent of methanol, acetonitrile, and isopropanol in a volume ratio of 3:6:1. The specific preparation process is as follows:

[0320] 100 μL of 11-deoxycortisol-d5, corticosterone-d8 and 18-hydroxycorticosterone-d4 standard internal standard stock solution, 500 μL of dehydroepiandrosterone-d6 standard internal standard stock solution and 1 mL of dehydroepiandrosterone sulfate-d6, 17-hydroxyprogesterone-d8, corticosterone-d8, androstenedione-d7, testosterone-d3, estrone-d4, estradiol-d5, estriol-d3, pregnenolone-d4, aldosterone-d7, progesterone-d9, 21-deoxycortisol-d8, 17-hydroxypregnenolone-d3, cortisol-d4 standard internal standard stock solution were respectively transferred into a 500 mL reagent bottle and washed with a third column. The solvent was diluted to volume to prepare a mixed internal standard working solution; wherein, the concentration of 11-deoxycortisol-d5, 17-hydroxyprogesterone-d8, corticosterone-d8, androstenedione-d7, testosterone-d3, corticosteroid-d8, estrone-d4, estradiol-d5, estriol-d3, pregnenolone-d4, aldosterone-d7, 18-hydroxycorticosterone-d4, progesterone-d9, 21-deoxycortisol-d8, 17-hydroxypregnenolone-d3, and cortisol-d4 was 200 ng / mL, the concentration of dehydroepiandrosterone-d6 was 1 μg / mL, and the concentration of dehydroepiandrosterone sulfate-d6 was 4 μg / mL.

[0321] After the internal standard solution is prepared, dispense 2 mL into brown penicillin bottles.

[0322] 5.3 Preparation of sample extract

[0323] Prepare a sample extract using one or more of n-hexane, methyl tert-butyl ether, and ethyl acetate. In a preferred embodiment, n-hexane is selected as the sample extract. The sample extract components are prepared by dispensing 120 mL of n-hexane into brown reagent bottles.

[0324] 5.4 Preparation of protein precipitant

[0325] The protein precipitant is prepared by mixing acetonitrile and methanol. Preferably, the protein precipitant is a mixed solvent having a volume ratio of acetonitrile to methanol of 9:1. The protein precipitant is dispensed into brown reagent bottles at 30 mL intervals to obtain the protein precipitant component.

[0326] 5.5. Preparation of Sample Enhancement Solution 1

[0327] Hydroxylamine hydrochloride is dissolved in a fourth solvent to prepare Sample Enhancement Solution 1, which is dispensed in 15 mL portions into brown reagent bottles. In a preferred embodiment, the concentration of the hydroxylamine hydrochloride solution is selected from any one of 0.5 mol / L, 1 mol / L, 2 mol / L, and 5 mol / L, and the fourth solvent is a 10% to 20% methanol aqueous solution or a 10% to 20% acetonitrile aqueous solution.

[0328] More preferably, the concentration of the hydroxylamine hydrochloride solution is 1 mol / L, and the fourth solvent is a 20vt% methanol aqueous solution. After the preparation is completed, 1 portion of the enhanced solution is dispensed into a brown reagent bottle for every 15 mL of the sample.

[0329] 5.6. Preparation of Sample Enhancement Solution 2

[0330] Prepare sample enhancement solution 2 by mixing 1,2-dimethylimidazole-5-sulfonyl chloride with the fifth solvent, and dispense 5 mL of the solution into brown penicillin bottles.

[0331] The concentration of 1,2-dimethylimidazole-5-sulfonyl chloride in the sample enhancement solution 2 is selected from any one of 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 5 mg / mL, and the fifth solvent is selected from acetone or ethyl acetate. More preferably, the concentration of the 1,2-dimethylimidazole-5-sulfonyl chloride solution is 1 mg / mL, and the fifth solvent is acetone. After preparation, each 5 mL is dispensed into a brown penicillin bottle.

[0332] 5.7 Preparation of sample conditioning solution

[0333] Add the weighed sodium bicarbonate and sodium carbonate respectively to an appropriate amount of deionized water and stir thoroughly until dissolved to prepare 0.1 mol / L sodium carbonate aqueous solution and 0.1 mol / L sodium bicarbonate aqueous solution, respectively. Mix the sodium carbonate solution and the sodium bicarbonate solution in a certain proportion to prepare the sample conditioning solution, and dispense 5 mL each into brown penicillin bottles.

[0334] Preferably, the volume ratio of 0.1 mol / L sodium carbonate aqueous solution to 0.1 mol / L sodium bicarbonate aqueous solution is 1:9 to 9:1, more preferably, the volume ratio of 0.1 mol / L sodium carbonate aqueous solution to 0.1 mol / L sodium bicarbonate aqueous solution is 6:4, and the sample conditioning solution is prepared and dispensed into brown penicillin bottles at 5 mL each.

[0335] 5.8 Preparation of Mobile Phase A and Mobile Phase B

[0336] Mix formic acid with water to prepare a 0.1vt% formic acid aqueous solution as mobile phase A; mix formic acid with methanol to prepare a methanol solution containing 0.1vt% formic acid as mobile phase B; mobile phase A and mobile phase B are respectively dispensed into reagent bottles at 0.5L each.

[0337] 5.9 Drying of test kit components

[0338] Six calibrators and two quality control products were dispensed into 5 mL brown vials at 0.5 mL each and placed in a freeze dryer for freeze drying. The internal standard solution, sample extract, protein precipitant, sample enhancement solution 1, sample enhancement solution 2, sample conditioning solution, mobile phase A, and mobile phase B did not need to be dried; the liquid components were sufficient.

[0339] In a preferred embodiment, the calibrators and quality control products are freeze-dried using the same freeze-drying process, which includes a pre-freezing stage, sublimation drying, and desorption drying in sequence; the operating conditions of the pre-freezing stage are shown in Table 1; the operating conditions of the sublimation drying are shown in Table 2; and the operating conditions of the desorption drying are shown in Table 3.

[0340] In a more preferred embodiment, the calibrators and quality control products are freeze-dried using the same freeze-drying process, which includes a pre-freezing stage, sublimation drying, and desorption drying in sequence; the operating conditions of the pre-freezing stage are shown in Table 4; the operating conditions of the sublimation drying are shown in Table 5; and the operating conditions of the desorption drying are shown in Table 6.

[0341] The 6 freeze-dried calibrators and 2 quality control products were assembled with the internal standard solution, sample extract, protein precipitant, sample enhancement solution 1, sample enhancement solution 2, sample adjustment solution, mobile phase A, and mobile phase B, and stored at -20°C to obtain a steroid hormone detection kit.

[0342] 6. Sample processing process

[0343] (1) Remove the kit (each reagent in the kit is prepared using the above-mentioned optimal parameters) from -20°C, return it to room temperature, and then remove the components in the kit;

[0344] (2) Add 0.5 mL of ultrapure water to each of the six calibrators and two quality control bottles, shake for 30 seconds, and set aside.

[0345] (3) Take 0.3 mL of each calibrator, each quality control product, and the serum sample to be tested and place them in a 2 mL centrifuge tube. Add 20 μL of internal standard solution to each sample and shake for 1 min.

[0346] (4) Add 0.3 mL of protein precipitant and 1.2 mL of sample extract to each centrifuge tube, vortex and oscillate for 3 min at 12000 rpm. -1 Extraction was performed by centrifugation for 5 min;

[0347] (5) The supernatant was placed in a new 2 mL centrifuge tube and blown dry with nitrogen at 40 °C;

[0348] (6) Add 80 μL of sample enhancement solution 1 to each dried centrifuge tube and incubate at 60°C with shaking for 15 min;

[0349] (7) Add 40 μL of sample enhancement solution 2 and 30 μL of sample conditioning solution to each centrifuge tube and incubate at 60°C with shaking for 15 min;

[0350] (8) Take out the incubated sample and spin at 12000 r·min-1 Centrifuge for 3 minutes, take 100 μL of the supernatant and place it in a liner tube in an injection vial or a 96-well plate;

[0351] (9) Place the injection vial liner or 96-well plate in the liquid chromatograph autosampler, and equilibrate the chromatographic column with mobile phase A and mobile phase B for LC-MS / MS detection;

[0352] The chromatographic conditions for LC-MS / MS detection are as follows:

[0353] Liquid chromatography conditions: Column: Fenome C8 ( 100×2.1mm, 2.6μm); column temperature: 40°C; mobile phase A: aqueous solution containing 0.1vt% formic acid; mobile phase B: methanol solution containing 0.1vt% formic acid; gradient elution; elution gradient: 0-0.5min, 35% mobile phase B; 0.5-5.0min, 35%-100% mobile phase B; 5.0-5.1min, 100%-35% mobile phase B; 5.1-7.0min, 35% mobile phase B; injection volume: 20μL; acquisition time: 7min.

[0354] Mass spectrometry conditions: an electrospray ion source was used, the acquisition mode was positive ion mode, the spray voltage was 3500 V, the sheath gas pressure was 40 Arb, the auxiliary gas pressure was 10 Arb, the evaporation temperature was 450°C, the ion transfer tube temperature was 350°C, the acquisition mode was multiple reaction monitoring (MRM), and the acquisition time was 7 min.

[0355] 7. Kit performance verification

[0356] The extracted ion chromatograms of 18 steroid hormones at the quantitative limit concentration are as follows Figure 6 shown.

[0357] The performance of the steroid hormone kit of the present invention was verified by designing experiments on linearity, limit of quantitation, precision, accuracy, etc. The verification results are shown in Tables 13 and 14. The chromatogram at the limit of quantitation concentration is shown in Table 13. Figure 6 .

[0358] (1) Linearity and quantification limit verification

[0359] The internal standard quantification method was adopted. TraceFinder software was used to establish a standard curve with the concentration ratio of the standard substance in the calibrator to the internal standard substance in the internal standard solution as the X-axis and the peak area ratio of the standard substance in the calibrator to the internal standard substance in the internal standard solution as the Y-axis. The sample at the lower limit of quantification concentration (calibrator C1) was detected. Each sample was tested 5 times. The linearity and quantification limit parameters of the kit, such as linear range, slope, correlation coefficient r, mean of the lower limit of quantification concentration, signal-to-noise ratio (S / N), coefficient of variation (CV%) and accuracy, were shown in Table 1. Figure 7The results showed that this method can achieve accurate detection at the lower limit of quantification (LLOQ) concentration and has a good linear relationship within the linear range.

[0360] (2) Precision verification

[0361] Stock solutions of 18 steroid hormones were added to normal human serum to prepare precision verification samples at high and low concentrations. The concentrations of the 18 steroid hormones were quantitatively determined using a steroid hormone kit using the isotope internal standard method. Each concentration was repeated five times, with one analytical batch per day for three consecutive days. The results of intra- and inter-assay precision are shown in Table 13.

[0362] Table 13 Precision results of 18 steroid hormone test kits (intra-batch and inter-batch precision)

[0363]

[0364]

[0365] (3) Accuracy verification

[0366] Working solutions of 18 steroid hormones were spiked into human serum. One of these solutions was not spiked with a standard, while the other three solutions were spiked with recovery verification standards at high, medium, and low concentrations. The concentrations of the 18 steroid hormones were quantitatively determined using the isotope internal standard method using a steroid hormone kit. The determination was repeated five times for each concentration, and the recoveries were calculated. The recovery results are shown in Table 14. The results showed that the spiked recoveries of the 18 steroid hormones in serum ranged from 68% to 121.6%.

[0367] Table 14 Accuracy (recovery) results of 18 steroid hormone kits

[0368]

[0369]

[0370]

[0371] The present invention provides a kit and method for the simultaneous detection of 18 steroid hormones based on dual derivatization technology. Numerous methods and approaches exist for implementing this technical solution. The foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A kit for the simultaneous detection of 18 steroid hormones based on double derivatization technology, characterized in that: The steroid hormones are 11-deoxycortisol, dehydroepiandrosterone, corticosterone, 18-hydroxycorticosterone, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, progesterone, cortisol, 21-deoxycortisol, aldosterone, testosterone, androstenedione, corticosterone, estrone, estradiol, estriol and dehydroepiandrosterone sulfate; The kit contains the following reagents: (1) Series of calibrators: The series of calibrators are freeze-dried products of a series of calibrator working solutions with six different concentration levels, and the series of calibrator working solutions are calibrator working solutions with six different concentration levels prepared by diluting a mixed working solution of steroid hormones of known concentration with a second solvent; The six concentration levels of the calibrator working solution are: The concentrations of pregnenolone, progesterone, estradiol, estriol, 21-deoxycortisol, aldosterone, and testosterone were the same, with the six concentrations being 0.05 ng / mL, 0.125 ng / mL, 0.25 ng / mL, 1.25 ng / mL, 5 ng / mL, and 10 ng / mL; The concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosterone, androstenedione, corticosterone, and dehydroepiandrosterone were the same, and the six concentrations were 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 2.5 ng / mL, 10 ng / mL, and 20 ng / mL; The concentrations of estrone and 18-hydroxycorticosterone were the same, and the six concentrations were 0.02 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, and 4 ng / mL; The six concentrations of dehydroepiandrosterone sulfate were: 50 ng / mL, 125 ng / mL, 250 ng / mL, 1250 ng / mL, 5000 ng / mL, and 10000 ng / mL; The six concentrations of cortisol were: 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 25 ng / mL, 100 ng / mL, and 200 ng / mL; (2) Series of quality control products: The series of quality control products are freeze-dried products of a series of quality control product working solutions with two different concentration levels, and the series of quality control product working solutions are quality control product working solutions with two different concentration levels prepared by diluting a steroid hormone mixed working solution of known concentration with a second solvent; The two concentration levels of the quality control working solution are: The concentrations of pregnenolone, progesterone, estradiol, estriol, 21-deoxycortisol, aldosterone, and testosterone were the same, with two concentration points of 0.2 ng / mL and 7.5 ng / mL, respectively; The concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosterone, androstenedione, corticosterone, and dehydroepiandrosterone were the same, with two concentration points of 0.4 ng / mL and 15 ng / mL, respectively; The concentrations of estrone and 18-hydroxycorticosterone were the same, with two concentration points of 0.08 ng / mL and 3 ng / mL, respectively; The two concentration points of dehydroepiandrosterone sulfate were 200 ng / mL and 7500 ng / mL; The two concentration points of cortisol are: 4 ng / mL and 150 ng / mL; (3) Internal standard solution: The internal standard solution is a mixture of various steroid hormone standard internal standard stock solutions of known concentrations, and is diluted with a third solvent to form an internal standard solution; In the internal standard solution, the concentrations of 11-deoxycortisol-d5, 17-hydroxyprogesterone-d8, corticosterone-d8, androstenedione-d7, testosterone-d3, corticosterone-d8, estrone-d4, estradiol-d5, estriol-d3, pregnenolone-d4, aldosterone-d7, 18-hydroxycorticosterone-d4, progesterone-d9, 21-deoxycortisol-d8, 17-hydroxypregnenolone-d3, and cortisol-d4 are 200 ng / mL, the concentration of dehydroepiandrosterone-d6 is 1 μg / mL, and the concentration of dehydroepiandrosterone sulfate-d6 is 4 μg / mL; (4) Sample extraction liquid: any one or a combination of n-hexane, methyl tert-butyl ether and ethyl acetate; (5) Protein precipitant: a mixed solvent of acetonitrile and methanol in any volume ratio; (6) Sample conditioning solution: a mixed solution of 0.1 mol / L sodium carbonate aqueous solution and 0.1 mol / L sodium bicarbonate aqueous solution in a volume ratio of 1:9 to 9:1; (7) Sample enhancement solution 1: hydroxylamine hydrochloride solution, the solvent in the solution is 10% to 20% methanol aqueous solution or 10% to 20% acetonitrile aqueous solution, and the concentration of hydroxylamine hydrochloride in the solution is 0.5 mol / L to 5 mol / L; (8) Sample enhancement solution 2: 1,2-dimethylimidazole-5-sulfonyl chloride solution, the solvent in the solution is acetone or ethyl acetate, and the concentration of 1,2-dimethylimidazole-5-sulfonyl chloride in the solution is 0.2 mg / mL~5 mg / mL; (9) Mobile phase A: 0.1 vt% formic acid in water; (10) Mobile phase B: methanol solution containing 0.1 vt% formic acid or acetonitrile solution containing 0.1 vt% formic acid; The freeze-drying process of the calibrator and the quality control product includes a pre-freezing stage, sublimation drying and desorption drying in sequence; The operating conditions of the pre-freezing stage are as follows: ； The operating conditions of the sublimation drying are as follows: ； The operating conditions of the analytical drying are as follows: ； or, The freeze-drying process of the calibrator and the quality control product includes a pre-freezing stage, sublimation drying and desorption drying in sequence; The operating conditions of the pre-freezing stage are as follows: ； The operating conditions of the sublimation drying are as follows: ； The operating conditions of the analytical drying are as follows: 。 2. The kit according to claim 1, wherein The second solvent consists of bovine serum albumin, an antioxidant, a preservative, a buffer and water; the antioxidant is isoascorbic acid; the preservative is citric acid; the buffer is PBS buffer; and the third solvent is a mixed solvent of methanol, acetonitrile and isopropanol in any volume ratio.

3. The kit according to claim 1, wherein The second solvent consists of 1-5% bovine serum albumin, 0.05-2% isoascorbic acid, 0.05-2% citric acid, 10% 10×PBS buffer and deionized water.

4. The kit according to claim 1, wherein The second solvent consists of 1% bovine serum albumin, 0.1% isoascorbic acid, 0.1% citric acid, 10% 10×PBS buffer and deionized water.

5. The kit according to claim 1, wherein The third solvent is a mixed solvent of methanol, acetonitrile and isopropanol in a volume ratio of 3:6:

1.

6. The kit according to claim 1, wherein The steroid hormone mixed working solution of known concentration is prepared as follows: 11-deoxycortisol, dehydroepiandrosterone, cortisol, 18-hydroxycorticosterone, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, progesterone, cortisol, 21-deoxycortisol, aldosterone, testosterone, androstenedione, corticosterone, estrone, estradiol, and estriol were accurately weighed and dissolved in the first solvent to prepare 1 mg / mL standard stock solutions; dehydroepiandrosterone sulfate was accurately weighed and dissolved in the first solvent to prepare 2 mg / mL standard stock solutions; The above-mentioned standard stock solutions are then prepared into a steroid hormone mixed working solution using the first solvent; the concentrations of pregnenolone, progesterone, estradiol, estriol, 21-deoxycortisol, aldosterone, and testosterone in the steroid hormone mixed working solution are all 1 μg / mL; the concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, corticosteroid, androstenedione, corticosterone, and dehydroepiandrosterone are all 2 μg / mL; the concentrations of estrone and 18-hydroxycorticosterone are both 400 ng / mL; the concentration of dehydroepiandrosterone sulfate is 1 mg / mL, and the concentration of cortisol is 20 μg / mL.

7. The kit according to claim 1, wherein The known concentrations of various steroid hormone standard internal standard stock solutions were prepared as follows: 11-deoxycortisol-d5, dehydroepiandrosterone-d6, cortisol-d8, and 18-hydroxycortisol-d4 were accurately weighed and dissolved in the first solvent to prepare 1 mg / mL standard internal standard stock solutions; dehydroepiandrosterone sulfate-d6 was accurately weighed and dissolved in the first solvent to prepare 2 mg / mL standard internal standard stock solutions; pregnenolone-d4, 17-hydroxypregnenolone-d3, 17-hydroxyprogesterone-d8, progesterone-d9, cortisol-d4, 21-deoxycortisol-d8, aldosterone-d7, testosterone-d3, androstenedione-d7, corticosterone-d8 were accurately weighed. 8、 Estrone-d4, estradiol-d5, and estriol-d3 were dissolved in the first solvent and then prepared into 100 μg / mL standard internal standard stock solutions; The above-mentioned internal standard stock solutions are mixed and diluted with the third solvent to obtain the internal standard solution.

8. The kit according to claim 6 or 7, characterized in that The first solvent is a methanol solution containing 2,6-di-tert-butyl-4-methylphenol.

9. The kit according to claim 1, wherein The series of calibrator working solutions and the series of quality control working solutions are freeze-dried respectively to obtain the series of calibrators and the series of quality control products.

10. The kit according to claim 1, wherein The sample extraction solution is n-hexane; the protein precipitant is a mixed solvent of acetonitrile and methanol in a volume ratio of 9:1; the sample conditioning solution is a mixed solution of 0.1 mol / L sodium carbonate aqueous solution and 0.1 mol / L sodium bicarbonate aqueous solution in a volume ratio of 6:4; the sample enhancement solution 1 is a hydroxylamine hydrochloride solution, the solvent in the solution is a 20 vt% methanol aqueous solution, and the concentration of hydroxylamine hydrochloride in the solution is 1 mol / L; the sample enhancement solution 2 is a 1,2-dimethylimidazole-5-sulfonyl chloride solution, the solvent in the solution is acetone, and the concentration of 1,2-dimethylimidazole-5-sulfonyl chloride in the solution is 1 mg / mL; the mobile phase A is a 0.1 vt% formic acid aqueous solution; and the mobile phase B is a methanol solution containing 0.1 vt% formic acid. The freeze-drying process of the calibrator and the quality control product includes a pre-freezing stage, sublimation drying and desorption drying in sequence; The operating conditions of the pre-freezing stage are as follows: ； The operating conditions of the sublimation drying are as follows: ； The operating conditions of the analytical drying are as follows: 。 11. Use of the kit according to any one of claims 1 to 10 for the simultaneous detection of 18 steroid hormones in serum using high performance liquid chromatography tandem mass spectrometry.

12. The use according to claim 11, characterized in that The 18 steroid hormones are 11-deoxycortisol, dehydroepiandrosterone, corticosterone, 18-hydroxycorticosterone, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, progesterone, cortisol, 21-deoxycortisol, aldosterone, testosterone, androstenedione, corticosterone, estrone, estradiol, estriol and dehydroepiandrosterone sulfate; Before the determination of steroid hormones, the series of calibrators are reconstituted with ultrapure water according to the corresponding six concentration levels of the series of calibrator working solutions to obtain a series of calibrator working solutions; the series of quality control products are reconstituted with ultrapure water according to the corresponding two concentration levels of the series of quality control working solutions to obtain a series of quality control working solutions.

13. The use according to claim 12, characterized in that The steps for testing 18 steroid hormones in serum include: The internal standard solution was added to the series of calibrator solution, the series of quality control solution and the serum sample respectively; then a protein precipitant and a sample extract were added to each sample, centrifuged, and the supernatant was collected; the supernatant was blown dry, and after adding sample enhancement solution 1, an oximation reaction was carried out at 55-65° C.; the sample enhancement solution 2 and the sample adjustment solution were continuously added, and an esterification reaction was carried out at 55-65° C.; after the reaction was completed, the reaction solution was centrifuged, the supernatant was collected, and the supernatant was subjected to LC-MS / MS detection.

14. The use according to claim 13, characterized in that The chromatographic conditions for the LC-MS / MS detection are as follows: Liquid chromatography conditions: Column: Fenome C8, 100 Å, 100 × 2.1 mm, 2.6 μm, Fenome C18, 100 Å, 100 × 3 mm, 2.6 μm, or Waters BEH C18, 2.1 mm × 50 mm, 1.7 μm; Column temperature: 40°C; mobile phase A: aqueous solution containing 0.1 vt% formic acid; mobile phase B: methanol solution containing 0.1 vt% formic acid or acetonitrile solution containing 0.1 vt% formic acid; gradient elution; elution gradient: 0-0.5 min, mobile phase B 35%; 0.5-5.0 min, mobile phase B 35%-100%; 5.0-5.1 min, mobile phase B 100%-35%; 5.1-7.0 min, mobile phase B 35%; injection volume: 20 μL; acquisition time: 7 min; Mass spectrometry conditions: an electrospray ion source was used, the acquisition mode was positive ion mode, the spray voltage was 3500~5500V, the sheath gas pressure was 35~50 Arb; the auxiliary gas pressure was 6~10 Arb; the evaporation temperature was 450~500℃; the ion transfer tube temperature was 350~400℃, the acquisition mode was multiple reaction monitoring mode, and the acquisition time was 7min.

15. The use according to claim 13, characterized in that The chromatographic conditions for the LC-MS / MS detection are as follows: Liquid chromatography conditions: chromatographic column: Fenome C8, 100 Å, 100 × 2.1 mm, 2.6 μm; column temperature: 40°C; mobile phase A: aqueous solution containing 0.1 vt% formic acid; mobile phase B: methanol solution containing 0.1 vt% formic acid; gradient elution; elution gradient: 0–0.5 min, 35% mobile phase B; 0.5–5.0 min, 35%–100% mobile phase B; 5.0–5.1 min, 100%–35% mobile phase B; 5.1–7.0 min, 35% mobile phase B; injection volume: 20 μL; acquisition time: 7 min. The mass spectrometry chromatography conditions are as follows: an electrospray ion source is used, the acquisition mode is positive ion mode, the spray voltage is 3500 V, and the sheath gas pressure is 40 Arb; Auxiliary gas pressure: 10Arb; evaporation temperature: 450℃; ion transfer tube temperature: 350℃, acquisition mode: multiple reaction monitoring mode, acquisition time: 7 min.

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