A kit, a detection method and a sample pretreatment method for simultaneously detecting 90 kinds of steroid hormones and their metabolites in a urine sample

Abstract

The application provides a kit for simultaneously detecting 90 kinds of steroid hormones and metabolites thereof in a urine sample, a detection method and a sample pretreatment method, the kit comprising 90 kinds of steroid hormones and metabolites thereof mixed standard working solution, internal standard working solution, matrix, redissolving agent and eluent, and further comprising liquid-liquid extraction agent, stabilizer, acid solution, buffer and enzymatic hydrolysis solution. 2 The application is characterized in that the R of the standard curve is greater than 0.990. The application realizes the simultaneous detection of 90 kinds of steroid hormones and metabolites thereof in a urine sample, and has the advantages of simple and rapid operation, high throughput, real-time, accurate and effective monitoring of the level of steroid hormones and metabolites thereof in human urine, and wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical chemistry, environmental and health, and clinical detection technology, and particularly relates to a kit for simultaneously detecting 90 kinds of steroid hormones in human urine, a detection method and a sample pretreatment method. BACKGROUND

[0002] Steroid hormones are a class of lipophilic small molecule compounds, which play an indispensable important role in human metabolism, immune regulation and maintenance of life functions. However, if excessive intake, steroid hormones can cause endocrine disorders, and pose a potential risk to human health. Therefore, it is particularly important to accurately determine the concentration of various steroid hormones and to deeply understand the mutual relationship between them and other metabolites. The detection of steroid hormones in urine samples has higher sensitivity and specificity, and is suitable for regular and repeated monitoring. In addition, compared with blood samples, urine samples are more convenient to process, usually do not require complex derivatization treatment, and are more non-invasive, easy to operate and acceptable to recipients.

[0003] Although with the advancement of technology, various methods for simultaneously detecting steroid hormones have emerged, but limited by the sensitivity of the detector, mass spectrometry resolution, ion suppression effect, chromatographic separation capacity, sample preparation and data analysis complexity, the number of steroid hormones that can be simultaneously accurately quantified in the current methods is still quite limited. Most of the developed methods can only simultaneously detect a few steroid hormones at the required level. Jeon BW et al. (Jeon BW et al., Anal Bioanal Chem. 2011; 401(4): 1353-1363) used LC-MS / MS to quantify 44 steroid hormones in urine, but the coverage of the number of steroid hormones detected was still relatively small, which was mainly because under the condition of electrospray ionization (ESI), steroid hormones were difficult to ionize into [M+ H] + or [M-H] - ion, because their chemical structure lacks acidic or basic groups. In addition, due to the large span of the content of steroid hormones in urine, the linear range is wide, and the sensitivity requirement of the instrument is high, and the existing methods are still difficult to meet the requirements of accurate quantification. In order to meet the needs of clinical and environmental health research, it is urgent to develop high recovery rate pretreatment technology of multiple steroid hormones and high sensitivity and specificity analysis and detection methods. SUMMARY

[0004] Therefore, the application provides a kit, a detection method and a sample pretreatment method for simultaneously detecting 90 kinds of steroid hormones and metabolites thereof in a human urine sample.

[0005] To achieve the above object, the application provides the following technical scheme: the application provides a kit for simultaneously detecting 90 kinds of steroid hormones and metabolites thereof in a urine sample, the kit comprising 90 kinds of steroid hormones and metabolite mixed standard working solution, steroid hormone and metabolite isotope internal standard working solution, matrix, redissolving agent and eluent, characterized in that,

[0006] The kit further comprises liquid-liquid extraction agent, stabilizer, acid solution, buffer and enzyme solution,

[0007] The matrix is artificial urine from which steroid hormones are removed,

[0008] The redissolving agent is pure methanol,

[0009] The eluent comprises eluent A and eluent B, the eluent A is ultrapure water, and the eluent B is pure methanol,

[0010] The liquid-liquid extraction agent is a mixture of tert-butyl methyl ether / ethyl acetate in a volume ratio of 1:1,

[0011] The stabilizer is ascorbic acid,

[0012] The acid solution is acetic acid,

[0013] The buffer comprises buffer 1 and buffer 2, the buffer 1 is sodium acetate buffer, and the buffer 2 is sodium carbonate-sodium bicarbonate buffer,

[0014] The enzyme solution is β -glucuronidase / arylsulfatase,

[0015] The 90 kinds of steroid hormones and metabolites are estrone, 17 α -estradiol, 17 β -estradiol, 17 α -ethinyl estradiol, 2-methyl estradiol, 4-hydroxy estrone, 2-hydroxy estrone, 2-methyl estrone, 4-methyl estrone, estriol, epitestosterone, methyltestosterone, testosterone, androstenone, dehydroepiandrosterone, 1,4-androstadiene-3,17-dione, 11 β -hydroxyandrostenone, nandrolone, 19-hydroxyandrostenone, 5 β- Androstan-3, 17-dione, Testolactone, Adrenosterone, Boldenone, Clostebol, Nandrolone phenylpropionate, Nandrolone, Progesterone, Pregnenolone, 20 α - Hydroxyprogesterone, 20 β - Hydroxyprogesterone, 5 β - Hydroxyprogesterone, 5 α - Hydroxyprogesterone, 6 β - Hydroxyprogesterone, 11 α - Hydroxyprogesterone, 17 α - Hydroxyprogesterone, 4-Pregnene-17 α , 20 α - Diol-3-ketone, 4-Pregnene-17 α , 20 β - Diol-3-ketone, Dimethylpregnene, 1, 6-Bisdehydroprogesterone, 16 α - Methylprogesterone, 1-Dehydroprogesterone, 16-Dehydroprogesterone, Delta-6- progesterone, 6-Ketoprogesterone, 11 α - Hydroxyprogesterone acetate, Bromopregnenone, Norethynodrel, Norethisterone acetate, Ethynodiol, Medroxyprogesterone, Medroxyprogesterone acetate, 16, 17-Epoxyprogesterone, 17 α - Hydroxyprogesterone acetate, 17-Carboxyprogesterone, Cyproterone acetate, Melengestrol acetate, Dimethisterone acetate, Chlormadinone acetate, Chlormadinone acetate, Flurogestone acetate, Tetrahydrodeoxycortisol, Corticosterone, 21-Deoxycortisol tetrol, Cortisone, Methylprednisolone, α - Cortisone tetrol, β - Cortisone tetrol, 11-Deoxycortisol, Deoxycortisol, 3 α , 5 β - Tetrahydrocortisol, Rimegepant, Budesonide, Flunisolide, Triamcinolone, Fluocinolone, Desoxymethasone, Clobetasol, Flumethasone, Clobetasol propionate, Clocortolone propionate, Fluticasone propionate, 11-Oxofluticasone propionate, Hydrocortisone 17-valerate and Spironolactone.

[0016] Preferably, the 90 kinds of steroid hormones and metabolites mixed standard working solution is 90 kinds of steroid hormones and metabolites mixed standard methanol solution with concentrations of 1.0, 2.0, 5.0, 10.0, 20.0, 40.0, 50.0, 100.0, 200.0, 400.0 and 500.0 ng / mL respectively, the steroid hormone and metabolite isotope internal standard working solution is a steroid hormone and metabolite isotope internal standard methanol solution with a concentration of 50.0 ng / mL, the concentration of the buffer 1 is 1 mol / L, and the pH value is 5.5; the concentration of the buffer 2 is 0.05 mol / L, and the pH value is 10.14, the concentration of the stabilizer is 6% w / v, and the concentration of the enzyme solution is 0.5% w / v.β - the enzyme activity of glucuronidase is 10.8 U / mL, and the enzyme activity of arylsulfatase is 25 U / mL.

[0017] Preferably, the isotopic internal standard of the steroid hormones and metabolites thereof comprises at least one of the following: 13 C-labeled, deuterated markers, the isotopic internal standard of the steroid hormones and metabolites thereof comprises d1-estrone, d2-17 α - estradiol, d2-17 β - estradiol, d4-17 α - ethinyl estradiol, d5-2-methyl estradiol, d3-testosterone, d7-androstenedione, d2-dehydroepiandrosterone, d7-11 β - hydroxyandrostenedione, d3-bolandone, d3-trenbolone, d5-phenylpropionate nortestosterone, d3-testosterone propionate, d9-progesterone, d8-17 α - hydroxyprogesterone, d6-dydrogesterone, 13 C2-norgestimate, d6-methylnorgestrel, d7-etogestrel, d6-norethindrone acetate, d6-norgestimate, d3-mifepristone, d3-methyloxprogesterone, d 11 - 17-carboxyprogesterone, d3-cyproterone acetate, d3-melengestrol acetate, d4-cortisol, d8-cortisone, d8-cortodoxone, 13 C4-11-deoxycortisol, d8-deoxycortisol, d5-tetrahydrocortisone, d6-budesonide, d7-triamcinolone, 13 C3-fluorocinolone, d5-clobetasol, d3-chlorocinolone propionate, d5-fluticasone propionate, d3-spiolactone.

[0018] Preferably, the kit further comprises steroid hormone and metabolite quality control working solutions with concentrations of 10.0, 20.0 and 40.0 ng / mL.

[0019] The present application also provides a sample pretreatment method for simultaneously detecting 90 kinds of steroid hormones and metabolites in a urine sample, characterized in that the pretreatment method comprises treating the sample with an enzymatic solution and liquid-liquid extraction.

[0020] The reagents used include isotopic internal standard working solutions of steroid hormones and metabolites, reconstitution solvents, liquid-liquid extraction agents, stabilizers, acid solutions, buffers and enzymatic solutions.

[0021] The reconstitution solvent is pure methanol.

[0022] The liquid-liquid extraction agent is a mixture of tert-butyl methyl ether / ethyl acetate in a volume ratio of 1:1.

[0023] the stabilizer is ascorbic acid,

[0024] the acidic solution is acetic acid,

[0025] the buffers include buffer 1, which is a sodium acetate buffer, and buffer 2, which is a sodium carbonate-sodium bicarbonate buffer,

[0026] the enzymatic solution is β - glucuronidase / arylsulfatase,

[0027] the 90 kinds of steroid hormones and their metabolites are estrone, 17 α - estradiol, 17 β - estradiol, 17 α - ethinyl estradiol, 2-methylestradiol, 4-hydroxyestrone, 2-hydroxyestrone, 2-methyl estrone, 4-methyl estrone, estriol, epitiostanol, methyltestosterone, testosterone, androstenedione, dehydroepiandrosterone, 1,4-androstenedione, 11 β - hydroxyandrostenedione, nandrolone, 19-hydroxyandrostenedione, 5 β - androstan-3,17-dione, testolactone, adrenosterone, boldenone, trenbolone, nandrolone phenylpropionate, nandrolone, progesterone, pregnenolone, 20 α - hydroxypregnenolone, 20 β - hydroxypregnenolone, 5 β - dihydroprogesterone, 5 α - dihydroprogesterone, 6 β - hydroxyprogesterone, 11 α - hydroxyprogesterone, 17 α - hydroxyprogesterone, 4-pregnene-17 α , 20 α - diol-3-ketone, 4-pregnene-17 α , 20 β - diol-3-ketone, dydrogesterone, 1,6-bishydrogenated progesterone, 16 α - methylprogesterone, 1-dehydroprogesterone, 16-dehydroprogesterone, Delta-6-progesterone, 6-ketoprogesterone, 11 α - hydroxyprogesterone acetate, dimethylstilbestrol, norethisterone, norethisterone acetate, ethynodiol, methallenestril, pregnenodione, etogestrel, norgestimate, mifepristone, medroxyprogesterone, medroxyprogesterone acetate, 16,17-epoxypregnenolone, 17 α - hydroxyprogesterone acetate, 17-carboxyprogesterone, cyproterone acetate, melengestrol acetate, dimenestril acetate, chlormadinone acetate, fluoxymesterone acetate, tetrahydrodeoxycorticosterone, corticosterone, 21-deoxycortisol tetrol, cortisone, methylprednisolone, α - cortisone tetrol, β- cortol, 11-deoxycortisol, deoxycortisol, 3 α ,5 β - tetrahydrocortisol, rimexolone, budesonide, flunisolide, triamcinolone acetonide, fluocinolone acetonide, desoxymethasone, clobetasol, flumethasone, clobetasol propionate, clocortolone pivalate, fluticasone propionate, 11-oxofluricasone propionate, 17-valerate hydrocortisone and spironolactone,

[0028] The pretreatment method comprises:

[0029] S1) sample preparation

[0030] The sample is mixed with the isotopic internal standard working solution of the steroid hormone and its metabolites by vortexing, and the supernatant is obtained by centrifugation. The acid solution, the stabilizer, the buffer 1 and the enzyme solution are added to the supernatant, and the extraction solution is obtained after sufficient vortexing.

[0031] S2) sample extraction

[0032] The buffer 2 and the liquid-liquid extraction agent are added to the extraction solution obtained in the sample preparation step S1), and the mixture is vortexed thoroughly. The supernatant is obtained by centrifugation. The liquid-liquid extraction agent is added to the remaining extraction solution after removing the supernatant, and the mixture is vortexed thoroughly. The supernatant is obtained by centrifugation. The supernatants obtained in the two steps are combined to obtain the pre-measurement sample.

[0033] S3) concentration and constant volume

[0034] The pre-measurement sample obtained in the step S2) is concentrated to dryness by centrifugation, and then redissolved with the redissolving agent. The mixture is vortexed thoroughly, and the supernatant is obtained by centrifugation to obtain the measurement sample.

[0035] Preferably, the isotopic internal standard working solution of the steroid hormone and its metabolites is a steroid hormone and its metabolite isotopic internal standard methanol solution with a concentration of 50.0 ng / mL. The concentration of the buffer 1 is 1 mol / L, and the pH value is 5.5. The concentration of the buffer 2 is 0.05 mol / L, and the pH value is 10.14. The concentration of the stabilizer is 6% w / v. The enzyme activity of the glucuronidase in the enzyme solution is 10.8 U / mL, and the enzyme activity of the arylsulfatase is 25 U / mL. β

[0036] Preferably, the steroid hormone and its metabolite isotopic internal standard comprises at least one of the C label and the deuterium label of the steroid hormone. 13 C label, deuterium label, the steroid hormone and its metabolite isotopic internal standard comprise d1-estrone, d2-17 α - estradiol, d2-17 β - estradiol, d4-17 α ​- ethinyl estradiol, d5-2-methylestradiol, d3-testosterone, d7-androstenedione, d2-dehydroepiandrosterone, d7-11 β - hydroxyandrostenedione, d3-bolandione, d3-trenbolone, d5-phenylpropionate nortestosterone, d3-testosterone propionate, d9-progesterone, d8-17 α - hydroxyprogesterone, d6-dydrogesterone, 13 C2-norethindrone, d6-methylnorethindrone, d7-etogestrel, d6-norgestimate, d6-norgestrel, d3-mifepristone, d3-methyloxprogesterone, d 11 - 17-carboxyprogesterone, d3-cyproterone acetate, d3-melengestrol acetate, d4-cortisol, d8-cortisone, d8-cortisone, d8-deoxycorticosterone, 13 C4-11-deoxycorticosterone, d8-deoxycortisol, d5-tetrahydrocortisone, d6-budesonide, d7-triamcinolone, 13 C3-fluocinolone, d5-clobetasol, d3-chlorocinolone propionate, d5-fluticasone propionate, d3-spiolactone.

[0037] Preferably, in the S1) sample preparation step, the volume ratio of the sample, the isotopic internal standard working solution of steroid hormones and their metabolites, the acidic solution, the stabilizer, buffer 1 and the enzyme solution is 1000:50:5:10:100:60, the volume of the enzyme solution is 60 μL, and the pH value of the solution after adding the acidic solution and buffer 1 is 5-6;

[0038] In the S2) sample extraction step, the volume ratio of the sample, buffer 2 and liquid-liquid extraction agent is 1:1:4, the volume of the liquid-liquid extraction agent added twice is 2 mL, and the pH value of the solution after adding the buffer 2 is 9-10;

[0039] In the S1) and S2) steps, the centrifugal speed is 10000-11000 r / min, the temperature is 4 ℃, and the processing time is 10-12 min;

[0040] In the S3) concentration and constant volume step, the centrifugal speed is 1400-1500 r / min, the temperature is room temperature, and the processing time is 80 min.

[0041] The application also provides a method for simultaneously detecting 90 kinds of steroid hormones and their metabolites in a urine sample, characterized in that the detection method comprises the following steps:

[0042] SS1) Preparation of standard sample to be tested

[0043] The artificial urine sample without steroid hormones is mixed with 90 kinds of steroid hormones and metabolite mixed standard working solution with concentrations of 1.0, 2.0, 5.0, 10.0, 20.0, 40.0, 50.0, 100.0, 200.0, 400.0 and 500.0 ng / mL respectively, and the mixed solution is pretreated by the pretreatment method to obtain the test standard sample;

[0044] SS2) Preparation of the test sample

[0045] The test urine sample is pretreated by the pretreatment method to obtain the test sample;

[0046] SS3) Machine detection

[0047] The test standard sample and the test sample are detected and analyzed by using an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer, and the peak area of each steroid hormone and metabolite in the test standard sample or the test sample and the internal standard peak area are recorded;

[0048] SS4) Result analysis

[0049] The standard curve is drawn with the concentration of the 90 kinds of steroid hormones and metabolite mixed standard working solution as the abscissa (x) and "(target peak area / internal standard peak area) x steroid hormone isotope internal standard working solution concentration" as the ordinate (y), wherein the target peak area is the peak area of each steroid hormone and metabolite in the 90 kinds of steroid hormones and metabolite mixed standard working solution, and the internal standard peak area is the peak area of the steroid hormone and metabolite isotope in the steroid hormone isotope internal standard working solution. The content of each steroid and metabolite in the urine sample is obtained according to the standard curve and the ratio of the peak area of each steroid and metabolite in the test sample to the internal standard peak area;

[0050] In the step SS3), the ultra-high performance liquid chromatography conditions are as follows: the chromatographic column is Poroshell HPH-C18, the column temperature is 40°C, the injection volume is 2 μL, the eluent includes eluent A and eluent B, and the flow rate is 0.4 mL / min; the gradient is as follows: 0 min, 40% eluent B; 0-4 min, 40%-55% eluent B; 4-8 min, 55%-70% eluent B; 8-13 min, 70-85% eluent B; 13-14 min, 85%-100% eluent B; 14-17 min, 100% eluent B; 17-20 min, 100%-60% eluent B; 20 min, 60% eluent B, the eluent A is ultrapure water, and the eluent B is pure methanol;

[0051] The mass spectrometry conditions are: using an electrospray ion source, the detection mode is positive ion mode, the mass spectrometry detection uses a multiple reaction monitoring mode, the ion source temperature is 150 DEG C, the desolvation gas temperature is 450 DEG C, the desolvation gas flow is 900 L / Hr, the collision gas flow is 150 L / h, and the capillary voltage is 3.0 kV.

[0052] Preferably, the volume ratio of the matrix, the 90 kinds of steroid hormone and metabolite mixed standard working solution, and the steroid hormone and metabolite isotopic internal standard working solution in the step SS1) is 1000:50:50.

[0053] Preferably, before the step SS3), a step SS5) of preparing a test quality control product is further included, and the step SS5) includes using the above-mentioned pretreatment method to pretreat a mixed solution of a matrix and a steroid hormone and metabolite quality control product working solution to obtain the test quality control product, the concentration of the steroid hormone and metabolite quality control product working solution is 10.0, 20.0 and 40.0 ng / mL, and the volume ratio of the matrix, the steroid hormone and metabolite quality control product working solution, and the steroid hormone isotopic internal standard working solution is 1000:50:50; the test quality control product is used to verify the standard curve drawn in the step SS4.

[0054] The beneficial technical effects of the present application include but are not limited to:

[0055] (1) The present application uses an enzyme solution to enzymatically digest a urine sample, combines liquid-liquid extraction, and detects based on an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry technology. Steroid hormones mostly exist in the form of glucuronide or sulfate conjugates in urine. Some glucuronide or sulfate conjugates have no standard product and thus cannot be detected. In addition, the content of free steroid hormones is small and difficult to detect. The addition of an enzyme solution can help extract these target molecules, make them exist in the form of free steroid hormones, or increase the content of free steroid hormones, which is easier for subsequent analysis and detection. The present application uses a liquid-liquid extraction method to extract steroid hormones and metabolites in a urine sample, which does not need to go through a complex process of solid-phase extraction enrichment, derivatization, etc. The operation is simple, can simultaneously detect the content of multiple steroid hormones in a urine sample, reduces experimental cost, shortens analysis time, improves detection throughput, and has high accuracy, good repeatability, and good stability of detection results, which can meet the detection needs of steroid hormones in clinical practice. At the same time, for the problems of few detection types and low detection sensitivity in the prior art due to the difficulty of ionization of steroid hormones into [M+H] + or [M-H] - ions, etc., the present application adjusts the components such as acidic solutions and buffers used in the pretreatment method of the urine sample, so that the steroid hormones and metabolites form different adduct ions such as [M + H]+ , [M-H2O+H] + , [M+Na] + , [M-2H2O+H] + The use of the ions can expand the types and sensitivity of the simultaneously detected steroid hormones and metabolites.

[0056] (2) The detection method used by the kit is ultra-high performance liquid chromatography tandem mass spectrometry, which directly detects steroid hormones and metabolites in urine samples according to the inherent physical and chemical properties and mass-to-charge ratio of the analyte, and can eliminate the problems of low sensitivity, poor specificity and low accuracy caused by traditional indirect detection methods such as existing immune analysis methods.

[0057] (3) Through the optimization of the pretreatment step and the selection of chromatography and mass spectrometry conditions, two ion pairs are simultaneously used for qualitative analysis for each hormone, and the ion pair with higher signal response is used for quantitative analysis, so as to establish a positive ion mode collection method, and the relative retention time and qualitative ion pair are used as the qualitative basis, and the standard curve is quantified by using the matrix mixed steroid hormone and metabolite mixed standard working solution, R 2 All are greater than 0.990, and each steroid hormone standard curve presents good linearity in the linear range. The detection method provided by the present application can realize chromatography and mass spectrometry separation of 90 kinds of steroid hormones and metabolites, has strong specificity, wide coverage, can provide complete metabolite spectrum, and can explore the biological transformation pathway of steroid hormones.

[0058] (4) The detection method of the present application can simultaneously detect 90 kinds of steroid hormones and metabolites in human urine at one time, has good separation degree, stable baseline, has high recovery rate, high precision, high sensitivity, high stability, high selectivity, low detection limit, and the detection result is more real and reliable, wherein the recovery rate is 72.31%~129.8%, the detection limit is 0.0029~0.5023 ng / mL, the quantitative limit is 0.0098~1.6744 ng / mL, the matrix effect is 0.10%~46.56%, and the precision (RSD) is 0.56%~15.68%. Moreover, there is no research on simultaneously detecting so many kinds of steroid hormones in biological matrix at present, and the present application has made a breakthrough progress. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 The quantitative ion chromatogram (500 ng / mL) of 90 kinds of steroid hormones and metabolites in the 90 kinds of steroid hormones and metabolite mixed standard working solution of the present application is shown;

[0060] Figure 2The quantitative ion chromatogram of 10 estrogens in the 90 kinds of steroid hormones and metabolites mixed standard solution of the application (500 ng / mL) is shown;

[0061] Figure 3 The quantitative ion chromatogram of 16 androgens in the 90 kinds of steroid hormones and metabolites mixed standard solution of the application (500 ng / mL) is shown;

[0062] Figure 4 The quantitative ion chromatogram of 40 progestogens in the 90 kinds of steroid hormones and metabolites mixed standard solution of the application (500 ng / mL) is shown;

[0063] Figure 5 The quantitative ion chromatogram of 24 corticoids in the 90 kinds of steroid hormones and metabolites mixed standard solution of the application (500 ng / mL) is shown;

[0064] Figure 6 The quantitative ion chromatogram of 10 estrogens in the 90 kinds of steroid hormones and metabolites mixed standard solution of the application (50 ng / mL) is shown;

[0065] Figure 7 The quantitative ion chromatogram of 16 androgens in the 90 kinds of steroid hormones and metabolites mixed standard solution of the application (50 ng / mL) is shown;

[0066] Figure 8 The quantitative ion chromatogram of 40 progestogens in the 90 kinds of steroid hormones and metabolites mixed standard solution of the application (50 ng / mL) is shown;

[0067] Figure 9 The quantitative ion chromatogram of 24 corticoids in the 90 kinds of steroid hormones and metabolites mixed standard solution of the application (50 ng / mL) is shown;

[0068] Figure 10 The extraction recovery of different extraction methods and different extractants is shown;

[0069] Figure 11 The extraction recovery of different volumes of tert-butyl methyl ether / ethyl acetate mixed solution (1 / 1, V / V) is shown;

[0070] Figure 12 The extraction recovery of different extraction times is shown;

[0071] Figure 13 The extraction recovery of urine samples with different pH values is shown;

[0072] Figure 14 The extraction recovery of urine samples with different contents of βthe ratio of the peak area of the steroid hormones and their metabolites in the urine sample after glucuronidase / arylsulfatase treatment to the peak area of the corresponding steroid hormones and their metabolites in the steroid hormones and their metabolite mixed standard working solution;

[0073] Figure 15 The quantitative ion chromatograms of 2 μL and 5 μL injection volumes are shown.

[0074] Figure 16 The quantitative ion chromatograms of different mobile phases are shown. DETAILED DESCRIPTION

[0075] The technical solutions in the present application are further described below in combination with the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application. The methods used in the present application are conventional methods unless otherwise specified; the raw materials used are conventional commercially available products unless otherwise specified. For example, the names, abbreviations, brands and purities of the steroid hormone and its metabolite standards and the isotope internal standards used in the present application are shown in Tables 1 and 2 below, respectively.

[0076] Table 1 Names, abbreviations, brands and purities of steroid hormone and its metabolite standards

[0077]

[0078] Table 2 Names, abbreviations, brands and purities of steroid hormone and its metabolite isotope internal standards

[0079]

[0080] The present application provides a kit capable of simultaneously detecting 90 kinds of steroid hormones and their metabolites in a urine sample, which comprises 90 kinds of steroid hormone and its metabolite mixed standard working solution, steroid hormone and its metabolite isotope internal standard working solution, matrix, reconstitution solvent and eluent, and further comprises liquid-liquid extraction agent, stabilizer, acidic solution, buffer and enzymatic solution.

[0081] The application also provides a sample pretreatment method, including sample preparation, sample extraction, sample concentration and constant volume, the sample preparation includes adding an enzymatic solution to the sample for enzymolysis, most of the steroid hormones in urine exist in the form of glucuronide or sulfate conjugates, and the addition of the enzymatic solution can help extract these target molecules. In order to achieve the best enzymolysis effect, the inventors add an acidic solution and a buffer to the above sample to adjust the pH value of the solution, and add a stabilizer to prevent the degradation of steroid hormones during enzymolysis. The sample extraction uses a liquid-liquid extraction method, which is simple to operate and can extract as many steroid hormones and metabolites in the urine sample as possible. Finally, the extract is concentrated and constant volume is prepared with a redissolving agent to prepare a sample to be tested, and the specific pretreatment method includes:

[0082] S1) sample preparation

[0083] The urine sample is mixed with the steroid hormone and metabolite isotope internal standard working solution, and the supernatant is obtained by centrifugation. The acid solution, the stabilizer, the buffer 1, the enzymatic solution, and the stabilizer are added to the supernatant to adjust the pH value of the solution, and the pH value is preferably 5-6, and more preferably 5.5. The enzymolysis effect is optimized, and the extract is obtained after sufficient vortexing, wherein the volume ratio of the sample, the steroid hormone and metabolite isotope internal standard working solution, the acid solution, the stabilizer, the buffer 1, and the enzymatic solution is preferably 1000:50:5:10:100:60.

[0084] S2) sample extraction

[0085] The buffer 2 is added to the extract prepared in the sample preparation step S1) to stop the enzymolysis, and the pH value of the solution is preferably adjusted to 9-10, and more preferably 9, so that the subsequent extraction effect is optimized. After adding the liquid-liquid extraction agent and vortexing, the supernatant is obtained by centrifugation. The liquid-liquid extraction agent is added to the remaining extract after removing the supernatant, and the supernatant is obtained by centrifugation after sufficient vortexing. The two supernatants are combined to obtain a sample to be tested, wherein the volume ratio of the urine sample, the buffer 2, and the liquid-liquid extraction agent is preferably 1:1:4, and the volume of the extraction agent added twice is the same, preferably 2 mL.

[0086] S3) concentration and constant volume

[0087] The sample to be tested obtained in step S2) is centrifuged to dryness, redissolved with the redissolving agent, and the upper clear liquid is obtained by centrifugation after sufficient vortexing to obtain a sample to be tested.

[0088] The application also provides a method for simultaneously detecting 90 kinds of steroid hormones and metabolites thereof in a urine sample, which comprises sample pretreatment and sample detection, wherein the sample pretreatment is performed by using the pretreatment method described above, and the sample pretreatment method can be used not only for the pretreatment of the urine sample, but also for the pretreatment of a mixed solution of a steroid hormone and metabolite mixed standard working solution and a matrix, and a mixed solution of a quality control working solution and a matrix. In order to maintain a single variable, the application adds a matrix to the steroid hormone and metabolite mixed standard working solution and the quality control working solution, i.e., removes artificial urine containing steroid hormones, so as to reduce errors. In the following examples of the application, the matrix (AU-002) of Biopanda is taken as an example, which may also contain trace amounts of certain steroid hormones and metabolites, for example β E2, NDL, EE2, SP. The presence of trace amounts of the above substances in the matrix has no substantial influence on the detection method of the application. The pretreated sample is detected by using an ultra-high performance liquid chromatograph-tandem mass spectrometer, a series of concentrations of the 90 kinds of steroid hormone and metabolite mixed standard working solution after pretreatment are detected, a standard curve is established, and the detection value of the urine sample after pretreatment is determined by comparing the standard curve to determine the content of the steroid hormone in the urine sample. In addition, the method of the application can also include verifying the above standard curve by using the quality control working solution after pretreatment. The specific steps include:

[0089] Preparation of the sample to be detected

[0090] The urine sample is pretreated by using the pretreatment method described above to obtain the sample to be detected.

[0091] Preparation of the standard sample or quality control sample to be detected

[0092] The preparation of the standard sample or quality control sample to be detected is completely the same as the preparation method of the sample to be detected, except that the matrix is added to the steroid hormone and metabolite mixed standard working solution or the quality control working solution to prepare the sample for pretreatment.

[0093] Detection by using an ultra-high performance liquid chromatograph-triple quadrupole tandem mass spectrometer

[0094] The sample to be detected, the quality control sample to be detected, and the sample to be detected are detected and analyzed by using an ultra-high performance liquid chromatograph-triple quadrupole tandem mass spectrometer, and the peak area of each steroid hormone and metabolite in the sample to be detected, the quality control sample to be detected, or the sample to be detected and the internal standard peak area are recorded.

[0095] Result analysis

[0096] The standard curve is drawn with the concentration of the steroid hormone and metabolite mixed standard working solution as the horizontal coordinate (x) and "(target peak area / internal standard peak area) x concentration of steroid hormone isotope internal standard working solution" as the vertical coordinate (y), wherein the target peak area is the peak area of each steroid hormone and metabolite in the 90 kinds of steroid hormone and metabolite mixed standard working solution, the internal standard peak area is the peak area of the steroid hormone and metabolite isotope in the steroid hormone isotope internal standard working solution, and the content of each steroid and metabolite in the test quality control or sample is obtained according to the standard curve and the ratio of the peak area of each steroid and metabolite in the test quality control or sample to the internal standard peak area, wherein the test quality control is used to verify the standard curve. Example 1

[0097] This example illustrates the composition of the kit of the application, and verifies the accuracy of the sample pretreatment method of the application and the detection method of the application, including the linear range, matrix effect, recovery rate, precision, detection limit, and quantitative limit.

[0098] 1. Kit and composition thereof

[0099] (1) 90 kinds of steroid hormone mixed standard working solution: an appropriate amount of 90 kinds of steroid hormones and metabolites is weighed, as shown in Table 1, and a single standard stock solution with a mass concentration of 5000.0 μg / mL is prepared by using a methanol solution. A certain volume of the single standard stock solution is accurately removed to prepare a mixed standard stock solution with a concentration of 1.0 μg / mL, and then the mixed standard stock solution is diluted with methanol to prepare a 90 kinds of steroid hormone and metabolite mixed standard working solution with a concentration gradient of 1.0, 2.0, 5.0, 10.0, 20.0, 40.0, 50.0, 100.0, 200.0, 400.0, and 500.0 (ng / mL);

[0100] (2) Steroid hormone and metabolite isotope internal standard working solution: an appropriate amount of 40 kinds of steroid hormone isotope internal standards is weighed, as shown in Table 2, and a single standard stock solution with a mass concentration of 1000 μg / mL is prepared by using a methanol solution. A certain volume of the single standard stock solution is accurately removed to prepare a mixed standard stock solution with a concentration of 1000.0 ng / mL, and then the mixed standard stock solution is diluted with methanol to prepare a steroid hormone and metabolite isotope internal standard working solution with a concentration of 50.0 ng / mL;

[0101] (3) Quality control working solution: An appropriate amount of 90 kinds of steroid hormones and their metabolites, as shown in Table 1, were weighed, and a single standard stock solution with a mass concentration of 5000.0 μg / mL was prepared by using a methanol solution. A certain volume of the single standard stock solution was accurately removed, and a mixed standard stock solution with a concentration of 1.0 μg / mL was prepared, and then the mixed standard stock solution was diluted with methanol to obtain the quality control working solution, a total of 3 concentration points, and the steroid hormone concentration of each point was as follows: 10.0, 20.0, 40.0 (ng / mL);

[0102] (4) Matrix: Artificial urine without steroid hormones (Biopanda, AU-002, pH = 6.6 + / - 0.5);

[0103] (5) Acidic solution: Acetic acid (Guojia, analytical pure);

[0104] (6) Stabilizer: Ascorbic acid (McLin), the concentration of the stabilizer was 6% (w / v);

[0105] (7) Buffer: Buffer 1 was sodium acetate buffer (Shanghai Shangbao Biological Technology Co., Ltd.), the concentration was 1.0 mol / L, and the pH value was 5.5; buffer 2 was sodium carbonate-sodium bicarbonate buffer (Shanghai Shangbao Biological Technology Co., Ltd.), the concentration was 0.05 mol / L, and the pH value was 10.14;

[0106] (8) Enzymatic solution: β - Glucuronidase / arylsulfatase (Roche, enzyme activity was 10.8 U / mL (25℃, β - Glucuronidase) / 25 U / mL (25℃, arylsulfatase));

[0107] (9) Liquid-liquid extraction agent: Tert-butyl methyl ether (CNW, HPLC grade, 99.8%) / ethyl acetate (CNW, HPLC grade, 99.8%) mixed solution (1 / 1, v / v);

[0108] (10) Resolvent: Pure methanol solution (Thermo Fisher, HPLC grade methanol (gold standard));

[0109] (11) Eluent: Eluent A was pure water (Guangzhou Watsons Food and Beverage Co., Ltd., 105℃ high-temperature distilled water), and eluent B was pure methanol.

[0110] 2. Sample pretreatment method:

[0111] S1: Sample preparation

[0112] Accurately pipette 1 mL of the matrix, add 50 μL of 90 kinds of steroid hormones and their metabolites mixed standard working solution or steroid hormone and its metabolite quality control working solution of different concentrations respectively, then add 50 μL of 50.0 ng / mL steroid hormone and its metabolite isotope internal standard working solution and use a vortex mixer (Licheng Technology, Vortex-2500MT) to mix well; centrifuge at 10000 r / min at 4°C for 10 min to obtain the supernatant. Add 5 μL of acetic acid, 10 μL of ascorbic acid, 100 μL of sodium acetate buffer and 60 μL of β - Glucuronidase / arylsulfatase, after vortexing at 3000 r / min for 5 min, the extraction solution was obtained, the tube was sealed with sealing film, and the extraction was carried out at 37°C for 16 h in a water bath;

[0113] S2: sample extraction

[0114] After adding 1 mL of sodium carbonate-sodium bicarbonate buffer to the extraction solution obtained in the S1 step, vortex at 3000 r / min for 5 min to stop the enzymatic reaction. After adding 2 mL of t-butyl methyl ether / ethyl acetate mixture (1 / 1, V / V) to the above solution, vortex at 3000 r / min for 5 min, centrifuge at 10000 r / min at 4°C for 10 min, and take the supernatant; add 2 mL of t-butyl methyl ether / ethyl acetate mixture (1 / 1, V / V) to the remaining extraction solution after removing the supernatant, and vortex at 3000 r / min for 5 min, centrifuge at 10000 r / min at 4°C for 10 min, and take the supernatant, and combine the two supernatants to obtain the test standard or quality control.

[0115] S3: concentration and constant volume

[0116] After the test standard or quality control obtained in the S2 step was concentrated to dryness at 1500 r / min using a centrifugal concentrator (Beijing Jiaim Limited Company) at room temperature for 80 min, the pure methanol solution was added to constant volume to 100 μL, vortexed and centrifuged to take the upper clear liquid to obtain the test standard or quality control. The test standard or quality control was transferred to a 2 mL brown glass sample bottle with a 5x29 mm liner tube, the liner tube was a 250 μL glass insert with polymer legs, and the brown glass sample bottle was a 9 mm caliber brown screw thread automatic sample bottle.

[0117] 3. Detection method

[0118] The test standard and the test quality control were respectively introduced into the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (Japan Shimadzu, Xevo TQ-XS triple quadrupole mass spectrometer) for detection and analysis, and the chromatogram was recorded and the peak area of each type of steroid hormone and its metabolite in the test standard or quality control was detected. The quantitative ion chromatogram of the steroid hormone in the 500 ng / mL and 50 ng / mL test standard is shown in Figures 1-9 The numbers in the figure represent the steroid hormones and their metabolites, as shown in Table 3.

[0119] (1) Liquid chromatography condition setting: the detection instrument used was ultra-high performance liquid chromatography (ACQUITY UPLC / Xevo TQ-S, American Waters Company); the chromatographic column was Poroshell HPH-C18 (100 mm×2.1 mm, 2.7 µm, Agilent), the column temperature was 40 ℃, the mobile phase used eluent A pure water and eluent B pure methanol, the injection volume was 2 μL, and the flow rate was 0.4 mL / min; the gradient was as follows: 0 min, 40% eluent B; 0-4 min, 40%-55% eluent B; 4-8 min, 55%-70% eluent B; 8-13 min, 70-85% eluent B; 13-14 min, 85%-100% eluent B; 14-17 min, 100% eluent B; 17-20 min, 100%-60% eluent B; 20 min, 60% eluent B;

[0120] (2) Mass spectrometry condition setting: an electrospray ion source (ESI) was used, the detection mode was positive ion mode (ESI + ), mass spectrometry detection used multiple reaction monitoring mode (MRM), the ion source temperature was 150 ℃, the desolvation gas temperature was 450 ℃, the desolvation gas flow rate was 900 L / Hr, the collision gas flow rate was 150 L / h, and the capillary voltage was 3.0 kV. The quantitative ion pair, cone voltage (CV) and collision energy (CE) parameters of 90 steroid hormone standards are shown in Table 3 below, and the mass spectrometry analysis parameters of 40 steroid hormone isotopic internal standards are shown in Table 4 below.

[0121] Table 3 90 kinds of steroid hormone standards and their mass spectrometry analysis parameters

[0122]

[0123] Table 4. 40 kinds of steroid hormone isotopic internal standards and their mass spectrometry parameters

[0124]

[0125] 4. Result analysis:

[0126] (1) Linear range: According to the 90 kinds of steroid hormone mixed standard solution, the standard curve of the target was established, with the concentration of the steroid hormone mixed standard solution as the horizontal coordinate (x), and the "(target peak area / internal standard peak area) x internal standard working solution concentration" as the vertical coordinate (y). The retention time, standard curve and correlation of 90 kinds of steroid hormones are shown in Table 5. In order to ensure that the linear standard curve has at least 6 effective concentration points, 1.0, 2.0, 5.0, 10.0, 20.0, 40.0, 50.0, 100.0, 200.0, 400.0, 500.0 (ng / mL) concentrations of 90 kinds of steroid hormone mixed standard solution were added to the matrix respectively, so that the concentrations of each substance were 0.1, 0.2, 0.5, 1.0, 2.0, 4.0, 5.0, 10.0, 20.0, 40.0, 50.0 (ng / mL) respectively. Each concentration point was repeated at least 3 times. In this study, the regression coefficients (R 2 ) of the linear calibration curve of 90 kinds of target steroid hormones were all greater than 0.990, as shown in Table 5. It can be seen that each steroid hormone standard curve presents good linearity in the linear range, and the steroid hormones represented by each serial number in Table 5 are the same as those in Table 3.

[0127] (2) Matrix effect, recovery and precision: Blank and spiked groups were set in each batch of experiment to monitor the background interference, precision and authenticity of the analysis procedure. The quality control results of 90 kinds of steroid hormones are shown in Table 5, the matrix effect is in the range of 0.10%~46.56%, and the matrix effect of 63 substances is less than 20%. The detection data of the quality control samples are substituted into the standard curve, and the recovery is calculated by comparing each blank concentration and the spiked concentration. The recovery of 90 target substances at the 10.0 ng / mL spiked concentration level is 72.31%~129.8%, and the relative standard deviation (RSD) is 0.88%~15.68%; the recovery at the 20.0 ng / mL spiked concentration level is 82.27%~117.46%, and the RSD is 0.76%~14.99%; the recovery at the 40.0 ng / mL spiked concentration level is 81.29%~118.67%, and the RSD is 0.56%~13.19%. From the results, it can be seen that the matrix effect is within 50%, the recovery is in the range of 70%~130%, indicating that the detection system is stable and the results are reliable.

[0128] (3) Limit of detection and limit of quantification experiments

[0129] In view of the results shown above, β E2, NDL, EE2, SP, process blank, 7 process blanks, 3 standard deviations, LOD, 7 process blanks, 10 standard deviations, LOQ. The remaining 86 target substances have no blank interference, 1 ng / mL concentration, 7 times, 3 and 10 times standard deviations, respectively. According to the above algorithm, the method in urine is calculated as LOD 0.0029~0.5023 ng / mL, LOQ 0.0098~1.6744 ng / mL, indicating that trace amounts of steroid hormones in urine can be effectively detected and quantified.

[0130] The above results show that the detection method of the present application can accurately, reliably and simultaneously detect 90 kinds of steroid hormones in human urine at one time, with good separation degree, stable baseline, high precision, high sensitivity, high stability, high selectivity, low detection limit and more real and reliable detection results.

[0131] Table 5 90 kinds of steroid hormones determination method evaluation

[0132] Example 2

[0133] This example is the same as Example 1 except that it includes the pretreatment of the actual urine sample. The actual urine sample contains one female urine sample and one male urine sample, which were collected from a certain unit in Beijing. The urine samples were collected in 50 mL polypropylene tubes and stored at -80°C. The pretreatment method of the actual urine sample is as follows:

[0134] S1 Preparation of the actual urine sample

[0135] Accurately pipette 1 mL of the actual urine sample into 100 μL of 50.0 ng / mL steroid hormone and metabolite isotope internal standard working solution and vortex well. After centrifugation at 10000 r / min at 4°C for 10 min, the supernatant is obtained. Add 5 μL of acetic acid, 10 μL of ascorbic acid, 100 μL of sodium acetate buffer and 60 μL of β-glucuronidase / arylsulfatase, respectively. After vortexing at 3000 r / min for 5 min, the extraction solution is obtained. Seal the tube with a sealing film and incubate at 37°C for 16 h in a water bath. β -β-glucuronidase / arylsulfatase, vortex at 3000 r / min for 5 min, and then obtain the extraction solution. Seal the tube with a sealing film and incubate at 37°C for 16 h in a water bath.

[0136] S2 Extraction of the actual urine sample

[0137] After adding 500 μL of sodium carbonate-sodium bicarbonate buffer to the extraction solution obtained in step S1, vortex at 3000 r / min for 5 min. Add 2.0 mL of t-butyl methyl ether / ethyl acetate mixture (1 / 1, V / V) to the above solution, vortex at 3000 r / min for 5 min, and then centrifuge at 10000 r / min at 4°C for 10 min. Take the supernatant, add 2.0 mL of t-butyl methyl ether / ethyl acetate mixture (1 / 1, V / V) to the remaining extraction solution, vortex at 3000 r / min for 5 min, and then centrifuge at 10000 r / min at 4°C for 10 min. Take the supernatant, and combine the two supernatants to obtain the pre-test sample.

[0138] S3 Concentration and dilution

[0139] After centrifuging the pre-test sample obtained in step S2 at 1500 r / min at room temperature for 80 min until dry, add pure methanol solution to dilute to 100 μL. Vortex well, centrifuge and take the supernatant to obtain the test sample. Transfer the test sample to a 2 mL brown glass sample bottle containing a 5×29 mm liner tube.

[0140] The pretreated sample to be tested is then detected and analyzed using an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer. The liquid chromatography conditions and mass spectrometry conditions are the same as in Embodiment 1. The peak area ratio of each type of steroid hormone and its metabolite in the measured sample to be tested to the internal standard peak area is brought into the standard curve drawn in Embodiment 1 to calculate the concentration of each type of steroid hormone and its metabolite.

[0141] The measured concentrations of steroid hormones in different gender actual urine samples are shown in Table 6 below. A total of 31 types of steroid hormones are detected. Among them, α - The content of etiocholanolone in male and female urine samples is the highest.

[0142] Table 6: Detection of steroid hormones in urine samples of different genders

[0143] Embodiment 3

[0144] In this embodiment, the urine sample is derived from 4 human mixed urine. 1 mL of mixed urine is taken and 100 μL of 50.0 ng / mL 90 types of steroid hormones and their metabolite mixed standard working solution is added to ensure that all steroid hormones and their metabolites are contained in the urine sample. Three samples of the above urine are prepared, respectively, and are stored under different conditions and used for detection. The stability of the detection of each type of steroid hormone using the pretreatment and detection method of the present application under the following three conditions is compared.

[0145] Case 1: The amount of biological sample collected is usually not only used for one sample determination, so the stability after repeated freezing and thawing needs to be considered. The urine sample is placed in -80 ℃ freezing, and after being taken out and thawed, one third of the urine sample is pretreated and detected. The remaining urine sample is continuously placed in -80 ℃ freezing. After complete freezing, the remaining urine sample is taken out and thawed, half of the remaining urine sample is pretreated and detected, and the remaining urine sample is continuously placed in -80 ℃ freezing. After complete freezing, the remaining urine sample is taken out and thawed, pretreated and detected, and the RSD values of the three detections are compared; Case 2: The urine sample is pretreated, and the sample to be tested is aliquoted into 5 parts and stored in a -20 ℃ refrigerator. The sample is measured on the 1st, 2nd, 3rd, 5th and 7th day, respectively, and the RSD values of the five detections are compared; Case 3: Since the sample loading time of the 96-well plate is relatively long, it needs to be considered whether the sample will be degraded during the sample loading process. The sample is pretreated and immediately loaded for analysis, and then stored in the sample loading chamber at 4 ℃ for 30 h before repeating the sample loading for analysis. The RSD values of the two detections are compared. The pretreatment and detection of the sample are the same as in Embodiment 2.

[0146] The RSD results of the above three cases are shown in Table 7. The RSD values of the three cases are all <20%, indicating that the sample does not affect the determination by the pretreatment and detection method of the application after being repeatedly frozen and thawed for 3 times. Under the condition of repeated freezing and thawing, the pretreatment and detection method of the application has good stability, and the sample to be tested does not affect the results when stored at -20 ℃ for 1 week, and does not significantly degrade during sample injection, and the results are stable.

[0147] Table 7 Stability results

[0148] Example 4

[0149] This example compares the effects of different extraction methods or extractants on the detection results.

[0150] Preparation of A detection sample

[0151] Accurately take 1 mL of the matrix and add 100 μL of 50.0 ng / mL 90 kinds of steroid hormones and metabolites mixed standard working solution. After vortexing at 3000 r / min for 5 min, the detection sample is obtained. In the same way, 8 detection samples are prepared.

[0152] Extraction of B detection sample

[0153] Solid phase extraction

[0154] Take two detection samples in A and use Oasis HLB solid phase extraction column (Waters, Oasis PRiME HLB, 150 mg, 3 cc) and Sep-Pak Silica solid phase extraction column (Waters, SEP-PAK VAC PSA 3cc, 200 mg, 3 cc) for extraction treatment. The specific operation is as follows: first, use 2.0 mL of methanol and 2.0 mL of ultrapure water to pretreat and activate the solid phase extraction column, then pass the detection sample through the column, and use 2 mL of water / methanol mixture (95 / 5, v / v) to elute the impurities and dry under vacuum. Take the solid phase extraction column off the solid phase extraction device, elute the solid phase extraction column with 2 mL of methanol solution, and press the residual liquid in the column with an adapter combined needle tube to obtain the extraction liquid.

[0155] Liquid liquid extraction

[0156] Take six A in the detection sample respectively add 1.0 mL of tert-butyl methyl ether / ethyl acetate mixed solution (1 / 1, V / V), n-hexane, dichloromethane, ethyl acetate (EAC), tert-butyl methyl ether (MTBE), tert-butyl methyl ether / ethyl acetate mixed solution (MTBE / EAC, 5 / 1, V / V), 3000 r / min vortex 5 min, centrifugation at 10000 r / min, 4℃ 10 min after taking supernatant, the extraction liquid is obtained.

[0157] C concentration and constant volume

[0158] According to the same step as example 1, the extraction liquid obtained in step B is concentrated and constant volume respectively, and the sample to be tested is obtained.

[0159] Then the sample to be tested and 50.0 ng / mL 90 kinds of steroid hormones and metabolites mixed standard working solution are respectively detected and analyzed by ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry, and the liquid chromatography conditions and mass spectrometry conditions are the same as example 1. The peak area ratio of each kind of steroid hormone and its metabolite in the sample to be tested to the corresponding steroid hormone and metabolite in the mixed standard working solution is used to calculate the extraction recovery, that is, the extraction recovery = sample peak area / standard peak area × 100%, and the results are shown in Figure 10 Figure 10 The recovery rates of 11 kinds of steroid hormones and metabolites are exemplarily shown. It can be seen from Figure 10 that overall, the extraction recovery of solid phase extraction method is higher than that of liquid phase extraction method. In liquid phase extraction method, the extraction recovery stability of MTBE / EAC (1 / 1, v / v) used in the application as extractant is good and closer to 100%. Example 5

[0160] This embodiment compares the influence of different amounts of extractant on the detection results.

[0161] Preparation of A detection sample

[0162] Five detection samples are prepared by the same method as example 4.

[0163] Extraction of B detection sample

[0164] 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, 6.0 mL of tert-butyl methyl ether / ethyl acetate mixed solution (1 / 1, V / V) are added to the five detection samples obtained in step A respectively, 3000 r / min vortex 5 min, centrifugation at 10000 r / min, 4℃ 10 min after taking supernatant, the extraction liquid is obtained.

[0165] C concentration and constant volume

[0166] ​The extraction solution obtained in step B was concentrated and made up to volume according to the same procedure as in Example 1 to obtain the sample to be tested.

[0167] The sample to be tested and 50.0 ng / mL of the steroid hormone and metabolite mixed standard working solution were then detected and analyzed using an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer. The liquid chromatography conditions and mass spectrometry conditions were the same as in Example 1, and the extraction recovery was calculated (calculation method as in Example 4). The extraction recoveries after extraction of different volumes of tert-butyl methyl ether / ethyl acetate mixture (1 / 1, V / V) are shown in Table 2. Figure 11 Figure 11 The recoveries of 12 kinds of steroid hormones and metabolites are exemplarily shown. It can be seen from Table 3 that the extraction recoveries of the steroid hormones and metabolites are closer to 100% when 2 mL of the extractant is used. Figure 11 Example 6

[0168] This example compares the effects of different extraction times on the detection results.

[0169] Preparation of the sample A

[0170] Four samples were prepared by the same method as in Example 4.

[0171] Extraction of the urine sample B

[0172] 2.0 mL of tert-butyl methyl ether / ethyl acetate mixture (1 / 1, V / V) was added to the first sample obtained in step A, and then vortexed at 3000 r / min for 5 min. After centrifugation at 10000 r / min and 4°C for 10 min, the supernatant was taken and directly used as the extraction solution. The second sample was extracted in the same way as the first sample, and then the extraction operation was repeated once, i.e. 2.0 mL of tert-butyl methyl ether / ethyl acetate mixture (1 / 1, V / V) was added to the sample from which the supernatant had been removed, and then vortexed at 3000 r / min for 5 min. After centrifugation at 10000 r / min and 4°C for 10 min, the supernatant was taken, and the two supernatants were combined and used as the extraction solution. The third sample was extracted three times, and the three supernatants were combined and used as the extraction solution. The fourth sample was extracted four times, and the four supernatants were combined and used as the extraction solution.

[0173] Concentration and volume making C

[0174] The extraction solution obtained in step B was concentrated and made up to volume according to the same procedure as in Example 1 to obtain the sample to be tested.

[0175] ​​The test sample and 50.0 ng / mL 90 kinds of steroid hormones and metabolites of the mixed standard working solution are then detected and analyzed using an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer. The liquid chromatography conditions and mass spectrometry conditions are the same as in Example 1. The extraction recovery is calculated (calculation method is the same as in Example 4). The extraction recovery after different extraction times is shown in Table 2. Figure 12 Figure 12 The recovery rates of 12 kinds of steroid hormones and metabolites are exemplarily shown. It can be seen from Table 2 that the extraction recovery of steroid hormones and metabolites after 2 times of extraction by the extraction agent is closer to 100%. Figure 12 Example 7

[0176] This example compares the influence of different pH values of urine samples on the detection results.

[0177] Preparation of A detection sample

[0178] Accurately pipette 6 mL of the matrix into 600 μL of 50.0 ng / mL 90 kinds of steroid hormones and metabolites of the mixed standard working solution and vortex well. Then, divide the mixture into 6 equal parts. Add 200 μL of sodium acetate buffer, 20 μL of sodium acetate buffer, no buffer solution, 100 μL of sodium carbonate buffer, 200 μL of sodium carbonate buffer, and 500 μL of sodium carbonate buffer to the 6 parts of the mixture, respectively. Vortex at 3000 r / min for 5 min to obtain the detection sample. The pH values of the detection sample are 4-5, 5-6, 6.6, 7-8, 8-9, and 9-10, respectively.

[0179] Extraction of B detection sample

[0180] Add 2.0 mL of tert-butyl methyl ether / ethyl acetate mixture (1 / 1, V / V) to the detection sample, and vortex at 3000 r / min for 5 min. Centrifuge at 10000 r / min and 4°C for 10 min. Take the supernatant. Add 2.0 mL of tert-butyl methyl ether / ethyl acetate mixture (1 / 1, V / V) to the remaining detection sample after removing the supernatant, and vortex at 3000 r / min for 5 min. Centrifuge at 10000 r / min and 4°C for 10 min. Take the supernatant. Combine the two supernatants to obtain the extraction solution.

[0181] Concentration and constant volume of C

[0182] According to the same procedure as in Example 1, the extraction solution obtained in step B is concentrated and constant volume to obtain the test sample.

[0183] ​​The test sample and 50.0 ng / mL 90 kinds of steroid hormones and metabolites of the mixed standard working solution are detected and analyzed by using an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer. The liquid chromatography conditions and mass spectrometry conditions are the same as those in Example 1. The extraction recovery is calculated (the calculation method is the same as that in Example 4). The extraction recovery of the detection sample at different pH values after extraction by tert-butyl methyl ether / ethyl acetate mixed liquid (1 / 1, V / V) is shown in Table 8. Figure 13 Figure 13 The recovery rates of 11 kinds of steroid hormones and metabolites are exemplarily shown. It can be seen from Table 8 that the extraction recovery of the detection sample after extraction by the extraction agent is best at pH = 9-10. Figure 13 Example 8

[0184] This example compares the influence of different enzyme digestion conditions on the detection results.

[0185] Preparation of A detection sample

[0186] Accurately take 1 mL of mixed urine sample, which is a mixed urine sample of 4 men and women. After centrifugation at 10000 r / min and 4℃ for 10 min, the supernatant is obtained. Four detection samples are prepared in the same way. Then 5 μL of acetic acid, 10 μL of ascorbic acid, 100 μL of sodium acetate buffer and β - glucuronidase / arylsulfatase, vortex at 3000 r / min for 5 min, seal the tube with sealing film, and obtain the extraction solution after 16 h of water bath at 37℃. Among them, the volume of β - glucuronidase / arylsulfatase added in the four detection samples is 20 μL, 40 μL, 60 μL and 80 μL, respectively.

[0187] Extraction of B detection sample

[0188] After adding 500 μL of sodium carbonate-sodium bicarbonate buffer to the extraction solution obtained in step A, vortex at 3000 r / min for 5 min. After adding 2.0 mL of tert-butyl methyl ether / ethyl acetate mixed liquid (1 / 1, V / V) to the above solution, vortex at 3000 r / min for 5 min, and centrifuge at 10000 r / min and 4℃ for 10 min, take the supernatant. Add 2.0 mL of tert-butyl methyl ether / ethyl acetate mixed liquid (1 / 1, V / V) to the remaining extraction solution after removing the supernatant for secondary extraction, vortex at 3000 r / min for 5 min, and centrifuge at 10000 r / min and 4℃ for 10 min. Take the supernatant, and combine the supernatants of the two times to obtain the pre-test sample.

[0189] Concentration and constant volume of C ​​

[0190] The sample obtained in step B was concentrated and diluted to a fixed volume according to the same steps as in Example 1 to obtain the sample to be tested.

[0191] The test sample and a 50.0 ng / mL mixed standard working solution of 90 types of steroid hormones and their metabolites were then analyzed using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS / MS). The HPLC and mass spectrometry conditions were the same as in Example 1. β Plot the volume of glucuronidase / arylsulfatase added on the x-axis, and the ratio of the peak area of ​​steroid hormones and their metabolites in the test sample to the peak area of ​​the corresponding steroid hormones and their metabolites in the 50.0 ng / mL 90 steroid hormone and metabolite mixed standard working solution on the y-axis. Figure 14 As shown. Figure 14 The peak area ratios of six types of steroid hormones and their metabolites are shown as an example. Figure 14 It can be seen that using different amounts of β The levels of steroid hormones and their metabolites detected in urine samples treated with glucuronidase / arylsulfatase ranged from 20 µL to 80 µL, with a range of 40 µL to 60 µL. As the volume of enzyme added increased, the amount of steroid hormones and their metabolites extracted from the urine sample increased, reaching a plateau around 60 µL. To balance the detection effectiveness and cost-effectiveness of this invention, a dosage of 60 µL was used. Example 9

[0192] This embodiment compares the effects of different injection volumes on the detection results.

[0193] 100 μL of a 50.0 ng / mL mixed standard working solution of 90 types of steroid hormones and their metabolites was injected at 2 μL and 5 μL respectively using an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer (UHPLC-MS / MS). The HPLC and MS / MS conditions were the same as in Example 1. The quantitative ion chromatograms for the 2 μL and 5 μL injection volumes are shown below. Figure 15 As shown. Figure 15 The recovery rates of 11 types of steroid hormones and metabolites are illustrated exemplarily. Figure 15 It can be seen that when the injection volume is 2 μL, the peak shape of the quantitative ion chromatogram is good and the degree of symmetry is high. The higher the degree of peak symmetry, the purer the single component separated from the sample. When the peak is skewed or sharp, it usually indicates the presence of other components, which will reduce the sensitivity of the chromatographic peak, the accuracy of peak area integration, and the resolution between chromatographic peaks. Example 10

[0194] This embodiment compares the impact of the flow relative on the detection results.

[0195] Each 100 μL 50.0 ng / mL 90 kinds of steroid hormones and metabolites mixed standard working solution 2, the injection amount is selected 2 μL, using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry for detection analysis, the mobile phase is selected pure water / pure methanol and 0.1% (V / V) formic acid water / pure methanol respectively, other liquid chromatography conditions and mass spectrometry conditions are the same as example 1. Figure 16 Figure 16 The recovery rate of 7 kinds of steroid hormones and metabolites is exemplarily shown. Figure 16 It can be known that the response of each target substance is improved when pure water / pure methanol is used as the mobile phase.

[0196] It can be seen from the above examples that the kit, pretreatment method and detection method of the present application can simultaneously detect 90 kinds of steroid hormones and metabolites in human urine at one time, have good separation degree, stable baseline, high precision, high sensitivity, high stability, high selectivity, low detection limit and more real and reliable detection results. And the effect of the technical scheme of the present application reaches the best through the gradual exploration of the experimental conditions such as enzyme digestion condition, extraction condition and detection condition. The above examples are only used to illustrate the technical scheme of the present application but not limit it, although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical scheme of the present application can be modified or replaced equivalently without departing from the purpose and scope of the technical scheme of the present application, which should be covered in the scope of the claims of the present application.​

Claims

1. A method for simultaneous detection of 90 kinds of steroid hormones and their metabolites in a urine sample, characterized by, The detection method comprises the following steps: SS1) preparing a to-be-tested standard sample Using a steroid hormone-free artificial urine as a matrix, 90 kinds of steroid hormones and metabolite mixed standard working solutions with concentrations of 1.0, 2.0, 5.0, 10.0, 20.0, 40.0, 50.0, 100.0, 200.0, 400.0 and 500.0 ng / mL are mixed with the matrix at a volume ratio of 1000:50, and the mixed solution of the matrix and the 90 kinds of steroid hormones and metabolite mixed standard working solution is pretreated to obtain the to-be-tested standard sample; SS2) preparing a to-be-tested sample The to-be-tested urine sample is pretreated to obtain the to-be-tested sample; SS3) machine detection The to-be-tested standard sample and the to-be-tested sample are detected and analyzed by using an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer, and the peak area of each steroid hormone and metabolite in the to-be-tested standard sample or the to-be-tested sample and the peak area of the internal standard are recorded in the chromatogram; SS4) result analysis A standard curve is drawn with the concentration of the 90 kinds of steroid hormones and metabolite mixed standard working solution as the abscissa (x) and "(target peak area / internal standard peak area) x steroid hormone isotopic internal standard working solution concentration" as the ordinate (y), wherein the target peak area is the peak area of each steroid hormone and metabolite in the 90 kinds of steroid hormones and metabolite mixed standard working solution, and the internal standard peak area is the peak area of the steroid hormone and metabolite isotope in the steroid hormone isotopic internal standard working solution. The content of each steroid and metabolite in the urine sample is obtained according to the standard curve and the ratio of the peak area of each steroid and metabolite in the to-be-tested sample to the internal standard peak area; wherein, In the steps SS1) and SS2), the pretreatment comprises enzyme hydrolysis treatment and liquid-liquid extraction, comprising: S1) sample preparation The sample is mixed with a steroid hormone and metabolite isotope internal standard working solution, centrifuged to obtain supernatant, and then an acid solution, a stabilizer, buffer 1 and an enzyme solution are added to the supernatant, and after being fully vortexed, an extraction solution is obtained, wherein the steroid hormone and metabolite isotope internal standard working solution is a steroid hormone and metabolite isotope internal standard methanol solution with a concentration of 50.0 ng / mL, the acid solution is acetic acid, the stabilizer is ascorbic acid with a concentration of 6% w / v, the buffer 1 is sodium acetate buffer, and the enzyme solution is β - glucuronidase / arylsulfatase, wherein β - the enzyme activity of glucuronidase is 10.8 U / mL, the enzyme activity of arylsulfatase is 25 U / mL, and the volume ratio of the matrix or the to-be-tested urine sample, the steroid hormone and metabolite isotope internal standard working solution, the acid solution, the stabilizer, buffer 1, and the enzyme solution is 1000:50:5:10:100:60, the volume of the enzyme solution is 60 µL, and the pH value of the solution after adding the acid solution and buffer 1 is 5-6; S2) sample extraction The extraction solution prepared in the S1) sample preparation step is added with buffer 2 and liquid-liquid extraction agent, vortexed thoroughly, centrifuged to take the supernatant, and the remaining extraction solution after removing the supernatant is repeatedly added with liquid-liquid extraction agent, vortexed thoroughly, centrifuged to take the supernatant, and the two supernatants are combined to obtain a to-be-tested pre-sample, wherein the buffer 2 is a sodium carbonate-sodium bicarbonate buffer, the liquid-liquid extraction agent is a mixture of tert-butyl methyl ether / ethyl acetate with a volume ratio of 1:1, the volume ratio of the sample, the buffer 2 and the liquid-liquid extraction agent is 1:1:4, the volume of the liquid-liquid extraction agent added twice is 2 mL, and the pH value of the solution after adding the buffer 2 is 9-10; S3) concentration and constant volume The to-be-tested pre-sample obtained in the S2) step is centrifuged to dryness, redissolved with a redissolving agent, vortexed thoroughly, centrifuged to take the upper clear liquid, and the to-be-tested standard sample or the to-be-tested sample is obtained, wherein the redissolving agent is pure methanol; In the step SS3), the ultra-high performance liquid chromatography conditions are as follows: the chromatographic column is Poroshell HPH-C18, the column temperature is 40 DEG C, the injection volume is 2 mu L, the eluent includes eluent A and eluent B, and the flow rate is 0.4 mL / min; the gradient is as follows: 0 min, 40% eluent B; 0-4 min, 40%-55% eluent B; 4-8 min, 55%-70% eluent B; 8-13 min, 70-85% eluent B; 13-14 min, 85%-100% eluent B; 14-17 min, 100% eluent B; 17-20 min, 100%-60% eluent B; 20 min, 60% eluent B, the eluent A is ultrapure water, and the eluent B is pure methanol; The mass spectrometry conditions are as follows: an electrospray ion source is used, the detection mode is a positive ion mode, mass spectrometry detection uses a multiple reaction monitoring mode, the ion source temperature is 150 DEG C, the desolvation gas temperature is 450 DEG C, the desolvation gas flow rate is 900 L / Hr, the collision gas flow rate is 150 L / h, and the capillary voltage is 3.0 kV; The 90 steroid hormones and their metabolites are estrone, 17 α -estradiol, 17 β -estradiol, 17 α -ethinyl estradiol, 2-methyl estradiol, 4-hydroxy estrone, 2-hydroxy estrone, 2-methyl estrone, 4-methyl estrone, estriol, epitiostanol, methyltestosterone, testosterone, androstenedione, dehydroepiandrosterone, 1,4-androstenedione, 11 β -hydroxyandrostenedione, nandrolone, 19-hydroxyandrostenedione, 5 β -androstan-3,17-dione, testolactone, adrenosterone, boldenone, trenbolone, nandrolone phenylpropionate, nandrolone decanoate, progesterone, pregnenolone, 20 α -hydroxypregnenolone, 20 β -hydroxypregnenolone, 5 β -dihydroprogesterone, 5 α -dihydroprogesterone, 6 β -hydroxyprogesterone, 11 α -hydroxyprogesterone, 17 α -hydroxyprogesterone, 4-pregnen-17 α ,20 α -diol-3-one, 4-pregnen-17 α ,20 β -diol-3-one, dydrogesterone, 1,6-bishydroxyprogesterone, 16 α -methylprogesterone, 1-dehydroprogesterone, 16-dehydroprogesterone, Delta-6- progesterone, 6-ketoprogesterone, 11 α -hydroxyprogesterone acetate, medroxyprogesterone, norethindrone, norethindrone acetate, norgestrel, megestrol, medroxyprogesterone acetate, 16,17-epoxymedroxyprogesterone, 17 α -hydroxyprogesterone acetate, 17-carboxyprogesterone, cyproterone acetate, melezestrol acetate, dimethisterone acetate, chlormadinone acetate, chlormadinone acetate, fluocortisone acetate, tetrahydrodeoxycortisol, corticosterone, 21-deoxycortisone tetrol, cortisone, methylprednisolone, α -cortisone tetrol, β -cortisone tetrol, 11-deoxycortisol, deoxycortisol, 3 α ,5 β -tetrahydrocortisol, rimexolone, budesonide, flunisolide, triamcinolone acetonide, fluocinolone acetonide, dexamethasone, clobetasol, flumethasone, clobetasol propionate, clocortolone, fluticasone propionate, 11-oxofluticasone propionate, 17- valerate hydrocortisone, and spironolactone.

2. The method of claim 1, wherein, The concentration of the buffer 1 is 1 mol / L, and the pH value is 5.5; the concentration of the buffer 2 is 0.05 mol / L, and the pH value is 10.

14.

3. The method of claim 1, wherein, The steroid hormones and metabolites thereof include at least one of 13 C-labeled, deuterated, or both, the steroid hormones and metabolites thereof include dl-estrone, d2-17 α -estradiol, d2-17 β -estradiol, d4-17 α -ethinyl estradiol, d5-2-methylestradiol, d3-testosterone, d7-androstenedione, d2-dehydroepiandrosterone, d7-11 β -hydroxyandrostenedione, d3-bolandione, d3-trenbolone, d5-phenylpropionate nortestosterone, d3-testosterone propionate, d9-progesterone, d8-17 α -hydroxyprogesterone, d6-dydrogesterone, 13 C2-norgestrel, d6-norgestimate, d6-etogestrel, d6-norethindrone acetate, d6-norgestimate, d3-mifepristone, d3-medroxyprogesterone, d 11 -17-carboxyprogesterone, d3-cyproterone acetate, d3-melengestrol acetate, d4-cortisol, d8-cortisone, d8-cortisol, d8-deoxycorticosterone, 13 C4-11-deoxycorticosterone, d8-deoxycortisol, d5-tetrahydrocortisol, d6-budesonide, d7-triamcinolone, 13 C3-fluorometholone, d5-clobetasol, d3-clocortolone propionate, d5-fluticasone propionate, d3-spiolactone.

4. The method of claim 1, wherein, In the steps S1) and S2), the centrifugal speed is 10000-11000 r / min, the temperature is 4 DEG C, and the treatment time is 10-12 min; In the step S3) of concentration and constant volume, the centrifugal speed is 1400-1500 r / min, the temperature is normal temperature, and the treatment time is 80 min.

5. The method according to any one of claims 1 to 4, characterized in that, Before the step SS3), a step SS5) of preparing a test quality control product is further included, the step SS5) includes that the mixed solution of the matrix and steroid hormone and metabolite quality control product working solution is subjected to the pretreatment, so that the test quality control product is obtained, the concentration of the steroid hormone and metabolite quality control product working solution is 10.0, 20.0 and 40.0 ng / mL, and the volume ratio of the matrix, the steroid hormone and metabolite quality control product working solution and the steroid hormone isotope internal standard working solution is 1000:50:50; the test quality control product is used for verifying the standard curve drawn in the step SS4.

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