A quantitative detection method for steroid hormones in human / animal body fluids and its application

Abstract

The invention discloses a quantitative detection method for 24 steroid hormones in human / animal plasma, serum, urine and other body fluids. The method is based on ultra-high performance liquid chromatography tandem mass spectrometry combined with online enzymolysis-solid phase extraction, which can realize one-time detection of the total amount of 24 steroid hormones in free and bound states, and the detection limit of the method can reach pg / mL, and the methodological validation results meet the 9101-analysis method validation guidelines. The detection method provided by the present invention can simultaneously and accurately quantitatively detect 24 steroid hormones in human / animal plasma, serum, urine and other body fluids, with high sensitivity, strong specificity, small sample size, short detection time, and improved method sensitivity, precision, detection efficiency and detection flux, low cost, and avoid waste of resources, providing efficient and powerful assistance for clinical diagnosis, monitoring and scientific research.

Description

Technical Field

[0001] The invention belongs to the technical field of clinical testing, and relates to a quantitative detection method and application of 24 types of steroid hormones in human / animal plasma, serum, urine and other body fluids. Background Art

[0002] Steroid hormones, also known as steroid hormones, are composed of four fused rings, and their precursor is cholesterol. Cholesterol undergoes a series of metabolic reactions to generate five major types of steroid hormones: progesterone, glucocorticoids, mineralocorticoids, estrogens, and androgens. The increase or decrease of human steroid hormones is closely related to some common clinical diseases, such as congenital adrenal hyperplasia, Cushing's syndrome, polycystic ovary syndrome, adrenal cortex insufficiency, etc. Monitoring the changes in the content of steroid hormones in the human body is one of the most important means for clinical testing and diagnosis.

[0003] Since there are many types of steroid hormones and their metabolites and their generally low content, they have similar skeleton structures and physical and chemical properties, and there are stereoisomers in these steroid hormones with the same structure, which brings great difficulties and challenges to the efficient and accurate detection of steroid hormones. Steroid hormones exist in the human body in free or bound forms. Hormones in different states play different functions and roles. Common bound steroid hormones mostly exist in the form of sulfate or bound to glucuronic acid. Due to the different binding sites, the number of isomers of bound steroid hormones produced has increased, which makes it difficult to directly detect bound steroid hormones. In clinical practice, the detection of steroid hormones mostly uses immunoassays to detect one or several items, and due to factors such as low sensitivity, poor specificity and low throughput of the detection method, there are often false positives or false negatives, time-consuming and labor-intensive situations. In addition, due to differences in reagents, methods, personnel, etc., the same sample has large differences in results between different laboratories, which has a great impact on the interpretation and treatment of related diseases. At present, bound steroid hormones have not been taken seriously and promoted, not only because of the interference of isomers in the direct detection of bound steroid hormones, but also because of the cumbersomeness and complexity of the operation. On the basis of ensuring the accurate quantification of free steroid hormones, more difficulties must be overcome to achieve further accurate quantification of bound steroid hormones. At present, there is no detection method that can quickly, accurately and efficiently quantify a variety of common bound and free steroid hormones. Summary of the invention

[0004] Liquid chromatography tandem mass spectrometry (LC-MS / MS) technology is one of the commonly used detection and analysis methods. Due to its advantages such as strong specificity, high sensitivity and fast analysis speed, it has been increasingly closely and deeply integrated with clinical diagnosis and monitoring in recent years, and has been favored by mass spectrometry scholars and medical researchers. Clinical mass spectrometry has also developed rapidly. Solid phase extraction technology (SPE) is a sample pretreatment technology developed in recent years. It is developed by combining liquid-solid extraction columns and liquid chromatography technology. It is mainly used for sample separation, purification and concentration. Compared with traditional liquid-liquid extraction methods, it can improve the recovery rate of analytes, more effectively separate analytes from interfering components, reduce sample pretreatment processes, and is simple to operate, time-saving and labor-saving. It is widely used in the fields of medicine, food, environment, commodity inspection, and chemical industry. In view of the shortcomings of the prior art mentioned above, the object of the present invention is to provide a method and application for quantitative detection of 24 steroid hormones in human / animal plasma, serum, urine and other body fluids, based on LC-MS, especially UPLC-MS / MS analysis platform, for simultaneous rapid and accurate quantitative detection of 24 steroid hormones in bound or free form in human / animal plasma, serum, urine and other body fluids.

[0005] To achieve the above-mentioned purpose and other related purposes, the present invention has developed a high-throughput, high-coverage, and highly sensitive targeted quantitative method for steroid hormones, which can accurately quantitatively analyze 24 steroid hormones in 13 minutes. The 24 steroid hormones cover a wide range, involving estrogen, progesterone, corticosteroids, androgens, and ecdysone, etc., which are more specific and practical than the existing clinical mass spectrometry detection technology. Compared with the traditional enzyme-linked immunosorbent assay and most of the existing clinical mass spectrometry detection technologies, the present invention quantifies the levels of multiple steroid hormones at the same time through one analysis, effectively saving manpower and material resources, improving the throughput, and meeting the detection requirements of most steroid hormones in clinical laboratories. Compared with the direct detection of bound steroid hormones, the present invention simplifies technical difficulties through online enzymatic hydrolysis-solid phase extraction technology. For any steroid hormone, the sum of its different bound states is directly detected, avoiding the interference of isomers of bound steroid hormones and the possible influence of complex matrices, thereby realizing the detection of the sum of any bound steroid hormone, which has a huge improvement in detection efficiency, throughput and convenience.

[0006] The present invention can detect free steroid hormones and obtain the content of bound steroid hormones by differential analysis by adding a mixed enzyme solution for online enzymolysis. In view of the current application status of clinical mass spectrometry technology in steroid hormone detection, it provides more portability, selectivity and comprehensiveness to better study the role of steroid hormones in different states in the life activities of humans / animals.

[0007] The present invention provides a quantitative detection method and application of 24 steroid hormones in human / animal body fluids, comprising the following steps:

[0008] Step 1) preparing a matrix mixed standard working solution: adding a plurality of steroid hormone standards to a first solvent for ultrasonic dissolution to obtain a steroid hormone single-label standard stock solution, adding a plurality of steroid hormone single-label standard stock solutions to a mixed solution of methanol and water for ultrasonic dissolution to obtain a steroid hormone mixed standard stock solution, then taking the steroid hormone mixed standard stock solution and adding it to a second solvent for ultrasonic dissolution to obtain mixed standard solutions of different concentrations, and finally using a 1% BSA solution to dilute the mixed standard solutions of each concentration to obtain matrix mixed standard working solutions of different concentrations;

[0009] Among them, the steroid hormones are: pregnenolone, 17α-hydroxypregnenolone, progesterone, 17α-hydroxyprogesterone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, estrone, estradiol, estriol, dihydrotestosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, cortisone, melatonin, aldosterone, androstenedione, cortisol, androstenediol, androsterone, epitestosterone, and 20-hydroxyecdysone.

[0010] The steps of adding the second solvent for ultrasonic dissolution to obtain mixed standard solutions of different concentrations are as follows: first, a high-concentration mixed standard solution (high-concentration standard curve) is prepared for further dilution to obtain a matrix mixed standard solution (the high-concentration standard curve is diluted with 1% BSA to obtain a low-concentration standard curve, which is used to quantify unknown samples); specifically, the specific concentrations of dehydroepiandrosterone sulfate in the mixed standard solutions of different concentrations are: 200000, 100000, 50000, , 20000, 10000, 5000, 2000, 100, 500, 200, 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, and 0.05ng / mL. The specific concentrations of the other 23 mixed standard solutions are 2000, 1000, 500, 200, 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, and 0.05ng / mL.

[0011] The first solvent is methanol, acetonitrile, DMSO, etc.; preferably, it is methanol or a mixed solvent of methanol and DMSO.

[0012] The volume ratio of methanol to DMSO is 1:1 to 1:9; preferably, 1:1.

[0013] The second solvent is methanol, acetonitrile, water, etc.; preferably, it is a mixed solvent of methanol and water.

[0014] The volume ratio of the mixed solution of methanol and water is 1:1 to 2:1; preferably, it is 1:1.

[0015] 2) Target extraction: The sample to be tested and the matrix mixed standard working solution of different concentrations are extracted by extractant 1 and extractant 2 using the organic reagent protein precipitation method to obtain the supernatant and protein precipitate respectively;

[0016] 3) Online enzymatic hydrolysis-solid phase extraction: The solid phase extraction column (Cleanert PEP, 2 mg) was activated with 200 μL methanol and then balanced with 200 μL water. The mixed solution of sulfatase and β-glucuronidase was loaded onto the solid phase extraction column, and the effluent was discarded to bond the sulfatase and β-glucuronidase to the filler of the solid phase extraction column. The supernatant obtained in step 2) was loaded onto the solid phase extraction column for a second time, the outflow valve was closed, and it was placed at 37°C for online enzymatic hydrolysis for 60 min. The outflow valve was slowly opened, the effluent was discarded, and the solid phase extraction column was eluted with eluent 1 and eluent 2 respectively, and the eluent was discarded. Finally, the target on the solid phase extraction column was eluted with eluent 1 and collected in a glass liner tube placed in a glass injection vial, and water was added to the initial proportion of the mobile phase and mixed to obtain the sample to be tested and the standard test product;

[0017] 4) Instrumental analysis: Ultra-high performance liquid chromatography-mass spectrometry is used to measure the samples to be tested and the standard test products in step 3), and the target analytes are identified based on the retention time and parent-daughter ion pair information. The chromatographic peak area of ​​the selected ion pairs of the target analytes is then quantified using the internal standard curve method to determine the content of various steroid hormones in the samples to be tested.

[0018] The steps of loading the enzyme mixture and performing online enzymolysis at 37° C. for 60 minutes are no longer performed when only the content of 24 free steroid hormones is detected. The content of 24 bound steroid hormones can be obtained by detecting the total content of 24 free and bound steroid hormones and the free content and taking the difference.

[0019] Step 1 of the present invention includes any one or more of the following conditions:

[0020] A1) The ultrasonic dissolution conditions are as follows: temperature: ice water bath; ultrasonic time: 1 to 5 min;

[0021] A2) the first solvent is ultrasonically dissolved in the following manner: estrone and 17α-hydroxypregnenolone are dissolved in a first solvent having a volume ratio of methanol or acetonitrile to DMSO of 1:1, and the rest are dissolved in pure methanol or pure acetonitrile;

[0022] A3) the volume ratio of the mixed solution of methanol and water is 1:1 to 2:1;

[0023] A4) the specific concentrations of dehydroepiandrosterone sulfate in the mixed standard solutions of different concentrations are: 200000, 100000, 50000, 20000, 10000, 5000, 2000, 1000, 500, 200, 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05 ng / mL, and the specific concentrations of the remaining 23 mixed standard solutions of different concentrations are 2000, 1000, 500, 200, 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05 ng / mL;

[0024] A5) The specific method of using 1% BSA solution to dilute the mixed standard solutions of various concentrations is as follows: prepare an aqueous solution with a mass fraction of 1% bovine serum albumin, and dilute the mixed standard solutions of various concentrations by 5 times.

[0025] Step 2 of the present invention includes any one or more of the following conditions:

[0026] B1) The organic reagent protein precipitation method is to take 200 μL of body fluid sample into a 1.5 mL PP centrifuge tube, first add 200 μL of extractant 1 and mix for 1 min, then add 200 μL of extractant 2 and mix for 1 min, and centrifuge at 12000 rpm at 3-5°C for 10 min;

[0027] B2) the seven isotopic internal standards contained in the extractant 1 are: estrone-d4, estradiol-d4, pregnenolone-d4, 17α-hydroxypregnenolone-d3, progesterone-d9, testosterone-d3, cortisol-d4, and the concentrations are all 50 ng / mL;

[0028] B3) the pH of the 2M acetic acid-sodium acetate buffer solution of the extractant 2 is about 5.2;

[0029] Step 3 of the present invention includes any one or more of the following conditions:

[0030] C1) The specific concentration of the mixed solution of sulfatase and β-glucuronidase is: β-glucuronidase: 200-300 U / mL, sulfatase: 600-800 U / mL, the solvent used is water, and the use volume is 80-120 μL; preferably, β-glucuronidase: 250 U / mL, sulfatase: 700 U / mL, the solvent used is water, and the use volume is 100 μL;

[0031] C2) the volume of the secondary loaded sample supernatant is 400-500 μL; preferably, 500 μL;

[0032] C3) the volume ratio of acetonitrile to water in the eluent 1 is 1:9, and the used volume is 150-250 μL, preferably 200 μL;

[0033] C4) the volume of the eluent 2 is 150-250 μL; preferably, 200 μL;

[0034] C5) the volume ratio of acetonitrile to methanol in the eluent 1 is 9:1, and the volume used is 30 to 50 μL; preferably, 35 μL;

[0035] C6) The enzyme mixture loading and online enzymatic hydrolysis at 37° C. for 60 min steps are no longer performed when only the content of the free 24 steroid hormones is detected.

[0036] In step 4 of the present invention, the liquid chromatography-mass spectrometry method is ultra-high performance liquid chromatography-tandem mass spectrometry;

[0037] The determination conditions of the ultra high performance liquid chromatography are:

[0038] Chromatographic column: C8 liquid chromatography column; column temperature: 45°C; injection volume: 5-20 μL; flow rate: 0.3 mL / min; mobile phase A is 0.5 mM ammonium fluoride aqueous solution; mobile phase B is methanol; analysis time is 13 min; gradient elution;

[0039] The mass spectrometry conditions are:

[0040] Ionization mode: electrospray ion source; ion source temperature is 500°C; curtain gas is 40psi; collision induced ionization parameter is medium; ion spray voltage is -4500 / 5500V; ion source Gas1: 60psi; Gas2: 55psi; monitoring mode is multiple reaction monitoring MRM mode for primary / secondary mass spectrometry analysis.

[0041] The specific procedure of the gradient elution of the present invention is: 0-0.5min, the volume ratio of phase A:phase B is 55:45-55:45; 0.5-6min, the volume ratio of phase A:phase B is 55:45-45:55; 6-10min, the volume ratio of phase A:phase B is 45:55-5:95; 10-12min, the volume ratio of phase A:phase B is 5:95-5:95; 12-12.2min, the volume ratio of phase A:phase B is 5:95-55:45; 12.2-13min, the volume ratio of phase A:phase B is 55:45-55:45.

[0042] The steroid hormones described in the present invention are: pregnenolone, 17α-hydroxypregnenolone, progesterone, 17α-hydroxyprogesterone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, estrone, estradiol, estriol, dihydrotestosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, cortisone, melatonin, aldosterone, androstenedione, cortisol, androstenediol, androsterone, epitestosterone, and 20-hydroxyecdysone.

[0043] The concentration range of the steroid hormone standard product after reconstitution with the first solvent is 0.1-5 mg / mL; the first solvent used for reconstitution of the standard product is one or more of methanol, acetonitrile and DMSO.

[0044] The extractant 1 of the present invention is methanol containing 7 steroid hormone isotope internal standards;

[0045] The extractant 2 is a 0.5-2M acetic acid-sodium acetate buffer solution; preferably, 2M;

[0046] The eluent 1 is a mixed solution of water and acetonitrile, wherein the volume ratio of water to acetonitrile is 9:1;

[0047] The eluent 2 is n-hexane;

[0048] The eluent 1 is a mixed solution of methanol and acetonitrile, wherein the volume ratio of methanol to acetonitrile is 1:9.

[0049] The solid phase extraction column of the present invention is Cleanert PEP (2 mg).

[0050] The online enzymolysis of the present invention uses a mixed solution of sulfatase and β-glucuronidase.

[0051] As described above, the quantitative detection method and application of 24 steroid hormones in human / animal plasma, serum, urine and other body fluids provided by the present invention can simultaneously and rapidly and accurately quantitatively detect 24 steroid hormones in human / animal plasma, serum, urine and other body fluids based on the UPLC-MS / MS analysis platform through online enzymatic hydrolysis combined with solid phase extraction technology, with high sensitivity and strong specificity, and for workers with certain mass spectrometry operation experience, it is easy to get started and can be operated by reading the instructions. The detection method requires a small amount of sample, optimizes the pretreatment and detection methods, has a short detection time, improves the sensitivity, precision, efficiency and detection flux of the method, is low in cost, avoids waste of resources, and provides efficient and powerful assistance for clinical diagnosis, monitoring and scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0053] Figure 1 Shown is a heat map of the test results of urine samples from 41 volunteers in Example 1 of the present invention.

[0054] Figure 2 Radar chart showing the test results of urine samples from 41 volunteers in Example 1 of the present invention

[0055] Figure 3 Shown is a standard curve diagram of some steroid hormones in the standard test product in Example 1 of the present invention, Figure 3 A is the standard curve of estrone, Figure 3 B is the standard curve of 17α-hydroxypregnenolone.

[0056] Figure 4 Shown is the extracted ion map of LC-MS / MS of some steroid hormones in a urine sample of a certain volunteer in Example 1 of the present invention.

[0057] Figure 5 , Figure 6 Shown is the extracted ion pattern of LC-MS / MS of 24 steroid hormones of the standard test sample s14 in Example 1 of the present invention.

[0058] Figure 7 , Figure 8 Shown is the extracted ion map of LC-MS / MS of 24 steroid hormones in a urine sample of a volunteer in Example 1 of the present invention. DETAILED DESCRIPTION

[0059] The invention is further described in detail with reference to the following specific embodiments and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0060] The first aspect of the present invention provides a method for quantitatively detecting 24 steroid hormones in human / animal plasma, serum, urine and other body fluids. The detection method includes 24 steroid hormones and 7 steroid hormone isotope internal standards as shown in Table 1 below.

[0061] Table 1

[0062]

[0063]

[0064] In the above detection method, the concentration range of the 24 steroid hormone standard products after ultrasonic reconstitution is 0.1-5 mg / mL, which are single-label standard stock solutions of 24 steroid hormones; the solvent used for reconstitution of the standard products is methanol or a mixed solution of methanol and DMSO in a volume ratio of 1:1; the conditions for ultrasonic dissolution are: temperature: ice water bath; ultrasonic time: 1-5 min.

[0065] In the above detection method, the mixed standard stock solution is obtained by mixing 24 steroid hormone single-label standard stock solutions with a water-methanol mixed solution; the volume ratio of water and methanol mixed solution used in the preparation of the mixed standard stock solution is 1:1.

[0066] In the above detection method, the mixed standard solution is obtained by diluting the mixed standard stock solution with a mixed solution of water and methanol, and the number is 21. The 21 mixed standard solutions are represented by S1, S2, S3, S4, ..., S18, S19, S20, and S21 respectively. The concentrations of various steroid hormones in S1 to S15 are arranged in order from small to large according to the concentration gradient: 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, and 20000 ng / mL. The concentrations of dehydroepiandrosterone sulfate in S16 to S21 are arranged in order from small to large according to the concentration gradient: 5000, 10000, 20000, 50000, 100000, and 200000 ng / mL; the volume ratio of water and methanol mixed solution used to dilute the mixed standard stock solution is 1:1.

[0067] In the above detection method, the matrix mixed standard working solution is obtained by diluting the mixed standard solution with a bovine serum albumin (BSA) solution with a mass fraction of 1%, and the number is 21. The 21 matrix mixed standard working solutions are represented by s1, s2, s3, s4, ..., s18, s19, s20, and s21 respectively. The concentrations of various steroid hormones in s1 to s15 are arranged in order from small to large according to the concentration gradient: 0.01, 0.02, 0.04, 0.1, 0.2, 0.4, 1, 2, 4, 10, 20, 40, 100, 200, and 400 ng / mL. The concentrations of dehydroepiandrosterone sulfate in s16 to s21 are arranged in order from small to large according to the concentration gradient: 1000, 2000, 4000, 10000, 20000, and 40000 ng / mL.

[0068] In the above detection method, the reagents also include extractant 1, extractant 2, eluent 1, eluent 2, and eluent 1. The extractant 1 is methanol containing 7 steroid hormone isotope internal standards. The 7 steroid hormone isotope internal standards are shown in Table 1 above, and the concentrations are all 50 ng / mL. The extractant 2 is a 2M acetic acid-sodium acetate buffer solution with a pH of about 5.2. The eluent 1 is a mixed solution of water and acetonitrile in a volume ratio of 9:1. The eluent 1 is n-hexane, and the eluent 1 is a mixed solution of methanol and acetonitrile in a volume ratio of 1:9.

[0069] The present invention provides a method and application for quantitatively detecting 24 steroid hormones in human / animal plasma, serum, urine and other body fluids, comprising the following steps:

[0070] 1) preparing matrix mixed standard working solutions: adding a plurality of steroid hormone standards to methanol or a mixed solvent of methanol and DMSO for ultrasonic dissolution to obtain a steroid hormone single standard stock solution, adding a plurality of steroid hormone single standard stock solutions to a mixed solution of methanol and water for ultrasonic dissolution to obtain a steroid hormone mixed standard stock solution, then taking the steroid hormone standard stock solution and adding a mixed solution of water and methanol for ultrasonic dissolution to obtain mixed standard solutions of different concentrations, and finally using 1% BSA solution to dilute the mixed standard solutions of each concentration to obtain matrix mixed standard working solutions of different concentrations;

[0071] 2) Target extraction: The sample to be tested and the matrix mixed standard working solution of different concentrations are extracted by extractant 1 and extractant 2 using the organic reagent protein precipitation method to obtain the supernatant and protein precipitate respectively;

[0072] 3) Online enzymatic hydrolysis-solid phase extraction: The solid phase extraction column (Cleanert PEP, 2 mg) was activated with 200 μL methanol and then balanced with 200 μL water. The mixed solution of sulfatase and β-glucuronidase was loaded onto the solid phase extraction column, and the effluent was discarded to bond the sulfatase and β-glucuronidase to the filler of the solid phase extraction column. 500 μL of the supernatant obtained in step 2) was loaded onto the solid phase extraction column for a second time, the outflow valve was closed, and it was placed at 37°C for online enzymatic hydrolysis for 60 min. The outflow valve was slowly opened, the effluent was discarded, and the solid phase extraction column was eluted with 200 μL of eluent 1 and eluent 2, respectively, and the eluent was discarded. Finally, 35 μL of eluent 1 was used to elute the target on the solid phase extraction column and collected in a glass liner tube placed in a glass injection vial, and water was added to the initial proportion of the mobile phase and mixed to obtain the sample to be tested and the standard test product;

[0073] 4) Instrumental analysis: Ultra-high performance liquid chromatography-mass spectrometry is used to measure the samples to be tested and the standard test products in step 3), and the target analytes are identified based on the retention time and parent-daughter ion pair information. The selected ion pair chromatographic peak area of ​​the target analytes is then quantified using the standard curve method to determine the content of various steroid hormones in the samples to be tested.

[0074] The steps of loading the enzyme mixture and performing online enzymolysis at 37° C. for 60 minutes are no longer performed when only the content of 24 free steroid hormones is detected. The content of 24 bound steroid hormones can be obtained by detecting the total content of 24 free and bound steroid hormones and the free content and taking the difference.

[0075] In the above step 1), BSA solutions with mass fractions of 0.5, 1, and 2 were tried. The test results of step 4) showed that the BSA solution with a mass fraction of 1% could better simulate the blank serum matrix without interfering with the peak and quantification of the target.

[0076] In the above step 2), the role of the extractant 2 is to provide a pH environment suitable for the enzymatic hydrolysis in step 3) and to dilute the supernatant produced in step 2) to avoid excessive loss of the target during the secondary loading in step 3).

[0077] In the above step 2), the extraction agent 1 was tried to be replaced with MTBE containing 7 isotope internal standards and acetonitrile containing 7 isotope internal standards. The detection results of step 4) showed that when the extraction agent 1 used methanol containing 7 isotope internal standards, it had a higher response intensity and signal-to-noise ratio for most target analytes.

[0078] In the above step 2), the organic reagent protein precipitation method is to take 200 μL of body fluid sample into a 1.5 mL PP centrifuge tube, first add 200 μL of extractant 1 and mix for 1 min, then add 200 μL of extractant 2 and mix for 1 min, and centrifuge at 12000 rpm at 3-5°C for 10 min.

[0079] In the above step 3), the eluent 1 was tried to be replaced with water or a mixed solution of water and acetonitrile in a volume ratio of 8:2. The test results of step 4) showed that when the extractant 1 used a mixed solution of water and acetonitrile in a volume ratio of 9:1, it had a higher response intensity and signal-to-noise ratio for most target analytes.

[0080] In the above step 3), the eluent 1 was tried to be replaced with a mixed solution of methanol and acetonitrile in a volume ratio of 1:1, or acetonitrile. The test results in step 4) showed that when the extractant 1 used methanol and acetonitrile in a volume ratio of 1:9, it had a higher response intensity and signal-to-noise ratio for most target analytes.

[0081] In the above step 3), the specific concentrations of the mixed solution of sulfatase and β-glucuronidase are: β-glucuronidase: 250 U / mL, sulfatase: 700 U / mL, the solvent used is water, and the volume used is 100 μL;

[0082] In the above step 3), the enzyme mixture loading and online enzymatic hydrolysis at 37° C. for 60 min are no longer performed when only the content of the free 24 steroid hormones is detected.

[0083] In the above step 4), in the ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS), the determination conditions of the ultra-high performance liquid chromatography (UPLC) are:

[0084] Chromatographic column: C8 liquid chromatography column; column temperature: 45°C; injection volume: 5-20 μL; flow rate: 0.3 mL / min; mobile phase A is 0.5 mM ammonium fluoride aqueous solution; mobile phase B is methanol; analysis time is 13 min; gradient elution;

[0085] In a further preferred embodiment, the determination conditions of the ultra high performance liquid chromatography (UPLC) are:

[0086] Chromatographic column: ACQUITYUPLC BEH C8 chromatographic column (column length 100 mm × inner diameter 2.1 mm, particle size 1.7 um); column temperature: 45°C; injection volume: 20 μL; flow rate: 0.3 mL / min; mobile phase A is 0.5 mM ammonium fluoride aqueous solution; mobile phase B is methanol; analysis time is 13 min; gradient elution.

[0087] In a further preferred embodiment, the specific procedure of the gradient elution is shown in Table 2, which is:

[0088] 0-0.5min, volume ratio of phase A:phase B was 55:45-55:45;

[0089] 0.5-6min, volume ratio of phase A:phase B is 55:45-45:55;

[0090] 6-10min, the volume ratio of phase A:phase B is 45:55-5:95;

[0091] 10-12min, volume ratio of phase A:phase B is 5:95-5:95;

[0092] 12-12.2min, volume ratio of phase A:phase B is 5:95-55:45;

[0093] 12.2-13min, the volume ratio of phase A:phase B is 55:45-55:45.

[0094] Table 2 Gradient elution parameters

[0095] Retention time (min) Flow rate (mL / min) Mobile phase A Mobile phase B 0 0.3 55 45 0.5 0.3 55 45 6 0.3 45 55 10 0.3 5 95 12 0.3 5 95 12.2 0.3 55 45 13 0.3 55 45

[0096] In the above step 4), in the ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS), the mass spectrometry (MS / MS) measurement conditions are:

[0097] Ionization mode: electrospray ion source; ion source temperature is 500°C; curtain gas is 40psi; collision induced ionization parameter is medium; ion spray voltage is -4500 / 5500V; ion source Gas1: 60psi; Gas2: 55psi; monitoring mode is multiple reaction monitoring MRM mode for primary / secondary mass spectrometry analysis.

[0098] The above mass spectrometry (MS / MS) includes a secondary mass spectrometry, in which the fragment ions of the primary mass spectrometry can be treated as parent ions and then fragmented and analyzed.

[0099] In one embodiment, in the ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS), the declustering voltage, collision voltage, parent ion, daughter ion, and retention time of 24 steroid hormones and 7 steroid hormone isotope internal standards are shown in Table 3.

[0100] Table 3

[0101]

[0102]

[0103] In the above step 4), the standard curve method comprises the following steps:

[0104] A1) treating a series of 24 steroid hormone mixed standard working solutions with different concentrations in step 2) and step 3) and then analyzing them by liquid chromatography-mass spectrometry to obtain the linear relationship between the chromatographic peak areas of the 24 steroid hormones and the peak areas of the 7 steroid hormone isotope internal standards and the corresponding concentrations, plotting the corresponding standard curves, and performing regression calculations using the weighted least squares method to obtain the linear regression equation of the analyte;

[0105] A2) The sample to be tested is subjected to UPLC-MS / MS analysis after being treated in step 2) and step 3), and the chromatographic peak areas of the 24 steroid hormones and the peak areas of the 7 steroid hormone isotope internal standards obtained are substituted into the regression equation of the standard working curve of the corresponding components in step A1) to calculate the concentration of the corresponding analyte in the sample to be tested.

[0106] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0107] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0108] The reagents or instruments used in the following examples without indicating the manufacturer are all conventional products that can be purchased from the market. For example, a vortex oscillator for sample mixing and a desktop high-speed refrigerated centrifuge for centrifugation. Among them, the ultra-high performance liquid chromatograph (UPLC) used for sample analysis and detection is produced by Shimadzu Corporation, and the mass spectrometer used for sample analysis and detection is a 6500 (QQQ / Qtrap) triple quadrupole series mass spectrometry system produced by Sciex Corporation.

[0109] Example 1

[0110] 1. Sample processing:

[0111] 41 tubes of urine were obtained from 41 volunteers as test samples. 200 μL of each test sample was taken into a 1.5 mL PP centrifuge tube. 200 μL of extractant 1 was first added and mixed for 1 min. Then 200 μL of extractant 2 was added and mixed for 1 min. The tube was centrifuged at 12000 rpm at 3-5°C for 10 min to obtain the supernatant and protein precipitate. The solid phase extraction column (Cleanert PEP, 2 mg) was activated with 200 μL methanol and then balanced with 200 μL water. 100 μL of a mixed solution of sulfatase and β-glucuronidase was loaded onto the solid phase extraction column, and the effluent was discarded to allow the sulfatase and β-glucuronidase to bond to the filler of the solid phase extraction column. 500 μL of the supernatant obtained after centrifugation was loaded onto the solid phase extraction column for a second time, the outflow valve was closed, and it was placed at 37°C for online enzymolysis for 60 minutes. The outflow valve was slowly opened, the effluent was discarded, and the solid phase extraction column was eluted with 200 μL of eluent 1 and eluent 2, respectively, and the eluent was discarded. Finally, 35 μL of eluent 1 was used to elute the target on the solid phase extraction column and collected in a glass liner tube placed in a glass injection vial, and 43 μL of water was added and mixed to obtain the sample to be tested.

[0112] The compositions of various solvents involved are shown in Table 4 below.

[0113] Table 4

[0114]

[0115]

[0116] *7 isotopic internal standards are: estrone-d4, estradiol-d4, pregnenolone-d4, 17α-hydroxypregnenolone-d3, progesterone-d9, testosterone-d3, cortisol-d4, and the concentration of each is 50ng / mL.

[0117] The samples to be tested were stored at -20℃ before being analyzed. The entire sample preparation process except the online enzymatic hydrolysis process was kept at low temperature.

[0118] 2. Preparation and treatment of matrix mixed standard working solution

[0119] According to the above step 1), a total of 21 matrix mixed standard working solutions of different concentrations are prepared, and the 21 matrix mixed standard working solutions are represented by s1, s2, s3, s4, ..., s18, s19, s20, and s21, respectively. The concentrations of various steroid hormones in s1 to s15 are arranged in order from small to large according to the concentration gradient: 0.01, 0.02, 0.04, 0.1, 0.2, 0.4, 1, 2, 4, 10, 20, 40, 100, 200, 400 ng / mL, and the concentrations of dehydroepiandrosterone sulfate in s16 to s21 are arranged in order from small to large according to the concentration gradient: 1000, 2000, 4000, 10000, 20000, 40000 ng / mL.

[0120] Take 200 μL of matrix mixed standard working solution into a 1.5 mL PP centrifuge tube, and the subsequent operations are consistent with the processing procedures of the samples to be tested to obtain the standard test product.

[0121] The standard test samples were stored at -20℃ before being analyzed on the machine. The entire sample preparation process except the online enzymatic hydrolysis process was kept at low temperature.

[0122] 3. Instrumental analysis

[0123] UPLC-MS / MS was used to measure the samples to be tested and the standard test products respectively. The characteristic peaks of the analytes and internal standards were identified according to the relative retention time and the parent-child ion pair information. Then, the analytes were quantified according to the standard curve to determine the content of each analyte in the sample to be tested.

[0124] In the UPLC-MS / MS analysis method, the UPLC measurement conditions are:

[0125] Chromatographic column: ACQUITYUPLC BEH C8 chromatographic column (column length 100 mm × inner diameter 2.1 mm, particle size 1.7 um); column temperature: 45°C; injection volume: 20 μL; flow rate: 0.3 mL / min; mobile phase A is 0.5 mM ammonium fluoride aqueous solution; mobile phase B is methanol; analysis time is 13 min; gradient elution.

[0126] The specific procedure of gradient elution is:

[0127] 0-0.5min, volume ratio of phase A:phase B was 55:45-55:45;

[0128] 0.5-6min, volume ratio of phase A:phase B is 55:45-45:55;

[0129] 6-10min, the volume ratio of phase A:phase B is 45:55-5:95;

[0130] 10-12min, volume ratio of phase A:phase B is 5:95-5:95;

[0131] 12-12.2min, volume ratio of phase A:phase B is 5:95-55:45;

[0132] 12.2-13min, the volume ratio of phase A:phase B is 55:45-55:45.

[0133] In the UPLC-MS / MS analysis method, the MS / MS measurement conditions are:

[0134] Ionization mode: electrospray ion source; ion source temperature is 500°C; curtain gas is 40psi; collision induced ionization parameter is medium; ion spray voltage is -4500 / 5500V; ion source Gas1: 60psi; Gas2: 55psi; monitoring mode is multiple reaction monitoring MRM mode for primary / secondary mass spectrometry analysis.

[0135] In the UPLC-MS / MS analysis method, the declustering voltage, collision voltage, parent ion, daughter ion, and retention time of the target analytes are shown in Table 3.

[0136] The test results of 41 volunteer urine samples are attached. Figure 1 and attached Figure 2 .

[0137] Example 2

[0138] In order to ensure the stability, sensitivity, accuracy and reliability of the detection method in the present invention, the present invention conducted a methodological investigation from aspects such as sensitivity, linear range, recovery rate and precision.

[0139] 1. Linearity and sensitivity investigation

[0140] The standard test samples s1 to s21 prepared in step 2 of Example 1 of the present invention were measured by UPLC-MS / MS method according to the detection conditions of step 3 of Example 1 of the present invention, and the linear range and detection limit of the detection method were investigated. The detection results are shown in Table 5 below. As can be seen from Table 5, the sensitivity of the detection method is high, as low as pg / mL level, and the linear method is wide, as wide as 3-5 orders of magnitude, which can meet the quantitative detection needs of 24 steroid hormones in human / animal plasma, serum, urine and other body fluids.

[0141] Table 5 Detection limit and linear range of target

[0142]

[0143]

[0144] Note: S / N: Signal / Noise.

[0145] 2. Recovery rate and precision investigation

[0146] The LQC, MQC and HQC were set and investigated by the sample spiking method. The concentrations of 24 steroid hormones corresponding to LQC, MQC and HQC were 2, 20 and 200 ng / mL, respectively. They were prepared by spiking commercial blank urine matrix purchased. The subsequent processing and detection process were consistent with steps 2 and 3 in Example 1. Three different personnel were arranged to carry out the experiment three times, and three batches of different data were obtained to calculate the inter-batch precision. One batch was selected to carry out three tests in one day to calculate the intra-day precision, and the test was carried out again on the second and third days to calculate the inter-day precision. The test samples were placed in a -20°C environment when not tested, and placed in the instrument sample tray during the test, and the temperature was set to 4°C.

[0147] The recovery and precision of the target analyte were calculated. The obtained recovery and method precision are shown in Table 6 below. The method recovery was between 80% and 120%, and the precision was within 20%. The method was stable and reliable.

[0148] Table 6 Method recovery and precision

[0149]

[0150]

[0151] Note: For results with multiple batches of data, take the average value of the multiple batches of data; n=3 for intra-day, inter-day and inter-batch precision.

[0152] In summary, the quantitative detection method and application of 24 steroid hormones in human / animal plasma, serum, urine and other body fluids provided by the present invention can simultaneously and rapidly and accurately quantitatively detect the content of 24 steroid hormones in human / animal plasma, serum, urine and other body fluids, with high sensitivity, strong specificity, small sample volume, short detection time, improved sensitivity, precision, efficiency and detection throughput of the method, low cost, and avoid waste of resources. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0153] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for quantitatively detecting steroid hormones in human or animal body fluids, characterized in that: The following steps are involved: Step 1, preparing a matrix mixed standard working solution: adding a plurality of steroid hormone standards to a first solvent for ultrasonic dissolution to obtain a steroid hormone single-label standard stock solution, adding a plurality of steroid hormone single-label standard stock solutions to a mixed solution of methanol and water for ultrasonic dissolution to obtain a steroid hormone mixed standard stock solution, then taking the steroid hormone mixed standard stock solution and adding a second solvent for ultrasonic dissolution to obtain mixed standard solutions of different concentrations, and finally using a 1% BSA solution to dilute the mixed standard solutions of each concentration to obtain matrix mixed standard working solutions of different concentrations; Step 2, target extraction: the sample to be tested and the matrix mixed standard working solution of different concentrations are extracted by extractant 1 and extractant 2 using an organic reagent protein precipitation method to obtain a supernatant and a protein precipitate respectively; Step 3, online enzymatic hydrolysis-solid phase extraction: the solid phase extraction column was activated with 200 µL of methanol and then balanced with 200 µL of water, the mixed solution of sulfatase and β-glucuronidase was loaded onto the solid phase extraction column, the effluent was discarded, the sulfatase and β-glucuronidase were bonded to the filler of the solid phase extraction column, the supernatant obtained in step 2 was reloaded onto the solid phase extraction column, the outflow valve was closed, and it was placed at 37°C for online enzymatic hydrolysis for 60 minutes, the outflow valve was slowly opened, the effluent was discarded, the solid phase extraction column was eluted with eluent 1 and eluent 2 respectively, the eluent was discarded, and finally the target on the solid phase extraction column was eluted with eluent 1 and collected into a glass liner tube placed in a glass injection vial, water was added to the initial proportion of the mobile phase and mixed, and the sample to be tested and the standard test product were obtained; Step 4, instrumental analysis: using ultra-high performance liquid chromatography-mass spectrometry to measure the sample to be tested and the standard test product in step 3, identifying the target analyte according to the retention time and the parent-daughter ion pair information, and then quantifying the chromatographic peak area of ​​the selected ion pair of the target analyte by the internal standard curve method to determine the content of various steroid hormones in the sample to be tested; The liquid chromatography-mass spectrometry method is an ultra-high performance liquid chromatography-tandem mass spectrometry method; The determination conditions of the ultra high performance liquid chromatography are: Chromatographic column: C8 liquid chromatography column; Column temperature: 45°C; injection volume: 5~20 µL; flow rate: 0.3 mL / min; mobile phase A is 0.5 mM ammonium fluoride aqueous solution; mobile phase B is methanol; analysis time is 13 min; gradient elution; The mass spectrometry conditions are: Ionization mode: electrospray ion source; ion source temperature: 500°C; curtain gas: 40 psi; collision induced ionization parameter: medium; ion spray voltage: -4500V for negative ion mode, 5500V for positive ion mode; ion source Gas1: 60 psi; Gas2: 55 psi; monitoring mode: multiple reaction monitoring (MRM) mode for primary and secondary mass spectrometry analysis; The steroid hormones are: pregnenolone, 17α-hydroxypregnenolone, progesterone, 17α-hydroxyprogesterone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, estrone, estradiol, estriol, dihydrotestosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, cortisone, melatonin, aldosterone, androstenedione, cortisol, androstenediol, androsterone, epitestosterone and 20-hydroxyecdysone.

2. The method for quantitative detection of steroid hormones in human or animal body fluids according to claim 1, characterized in that: Step 1 includes any one or more of the following conditions: A1) The ultrasonic dissolution conditions are as follows: temperature: ice water bath; ultrasonic time: 1-5 min; A2) The first solvent is ultrasonically dissolved in the following conditions: estrone and 17α-hydroxypregnenolone are dissolved in a first solvent having a volume ratio of methanol or acetonitrile to DMSO of 1:1, and the rest are dissolved in pure methanol or pure acetonitrile; the first solvent is one or more of methanol, acetonitrile, and DMSO; the second solvent is one or more of methanol, acetonitrile, and water; A3) the volume ratio of the mixed solution of methanol and water is 1:1 to 2:1; A4) The specific concentrations of dehydroepiandrosterone sulfate in the mixed standard solutions of different concentrations are: 200000, 100000, 50000, 20000, 10000, 5000, 2000, 1000, 500, 200, 100, 500, 200, 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1 and 0.05 ng / mL; the specific concentrations of the remaining 23 mixed standard solutions of different concentrations are 2000, 1000, 500, 200, 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1 and 0.05 ng / mL; A5) The specific method of using 1% BSA solution to dilute the mixed standard solutions of various concentrations is as follows: prepare an aqueous solution with a mass fraction of 1% bovine serum albumin, and dilute the mixed standard solutions of various concentrations 5 times.

3. The method for quantitative detection of steroid hormones in human or animal body fluids according to claim 1, characterized in that: Step 2 includes any one or more of the following conditions: B1) The organic reagent protein precipitation method is to take 200 µL of body fluid sample into a 1.5 mL PP centrifuge tube, first add 200 µL of extractant 1 and mix for 1 min, then add 200 µL of extractant 2 and mix for 1 min, and centrifuge at 12000 rpm at 3-5°C for 10 min; B2) The seven isotopic internal standards contained in the extractant 1 are: estrone-d4, estradiol-d4, pregnenolone-d4, 17α-hydroxypregnenolone-d3, progesterone-d9, testosterone-d3 and cortisol-d4, and the concentrations are all 50 ng / mL; B3) The pH value of the 2 M acetic acid-sodium acetate buffer solution of the extractant 2 is 5.

2.

4. The method for quantitative detection of steroid hormones in human or animal body fluids according to claim 1, characterized in that: Step 3 includes any one or more of the following conditions: C1) The specific concentrations of the mixed solution of sulfatase and β-glucuronidase are: β-glucuronidase: 200-300 U / mL, sulfatase: 600-800 U / mL, the solvent used is water, and the volume used is 80-120 µL; C2) The volume of the sample supernatant for the secondary loading is 400-500 µL; C3) the volume ratio of acetonitrile to water in the eluent 1 is 1:9, and the used volume is 150-250 μL; C4) the volume of the eluent 2 used is 150-250 µL; C5) the volume ratio of acetonitrile to methanol in the eluent 1 is 9:1, and the volume used is 30-50 μL; C6) The steps of loading the mixed solution of sulfatase and β-glucuronidase and online enzymatic hydrolysis at 37°C for 60 min are no longer performed when only the content of free 24 steroid hormones is detected.

5. The method for quantitative detection of steroid hormones in human or animal body fluids according to claim 1, characterized in that: The specific procedure of the gradient elution is: 0-0.5 min, the volume ratio of phase A: phase B is 55:45-55:45; 0.5-6 min, the volume ratio of phase A: phase B is 55:45-45:55; 6-10 min, the volume ratio of phase A: phase B is 45:55-5:95; 10-12 min, the volume ratio of phase A: phase B is 5:95-5:95; 12-12.2 min, the volume ratio of phase A: phase B is 5:95-55:45; 12.2-13 min, the volume ratio of phase A: phase B is 55:45-55:

45.

6. The method for quantitative detection of steroid hormones in human or animal body fluids according to claim 1, characterized in that: The concentration range of the steroid hormone standard after reconstitution with the first solvent is 0.1-5 mg / mL; the first solvent used for reconstitution of the standard is one or more of methanol, acetonitrile, and DMSO.

7. The method for quantitative detection of steroid hormones in human or animal body fluids according to claim 1, characterized in that: The extractant 1 is methanol containing 7 steroid hormone isotope internal standards; the extractant 2 is 0.5-2 M acetic acid-sodium acetate buffer solution; the eluent 1 is a mixed solution of water and acetonitrile; the eluent 2 is n-hexane, and the eluent 1 is a mixed solution of methanol and acetonitrile.

8. The method for quantitative detection of steroid hormones in human or animal body fluids according to claim 1, characterized in that: The solid phase extraction column is Cleanert PEP, and the weight of the filler is 2 mg.

9. The method for quantitative detection of steroid hormones in human or animal body fluids according to claim 1, characterized in that: The online enzymatic hydrolysis uses a mixed solution of sulfatase and β-glucuronidase.

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