Method for detecting urine steroid hormone metabolic profile by solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a method for detecting a urine steroid hormone metabolic profile by solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry. The method comprises the following steps: S1, preparing an internal standard stock solution and a target analyte standard stock solution; s2, pretreatment of a to-be-detected urine sample: S2.1, enzymolysis: taking the to-be-detected urine, adding the internal standard stock solution and the enzymolysis application solution, mixing, and incubating, so that uronide is fully hydrolyzed; s2.2, solid-phase extraction, separation and purification; s2.3, carrying out multi-step solvent distribution and redissolution; s2.4, filtering the reconstitution fluid through a filter membrane; s3, urine sample analysis: performing high performance liquid chromatography-triple quadrupole tandem mass spectrometry determination on the filtered reconstitution fluid; s4, drawing a standard curve; and S5, data analysis: performing internal standard method quantification on the analyte according to the ratio of the target peak area to the internal standard peak area. The detection method disclosed by the invention is high in accuracy, high in sensitivity and good in result reproducibility.

Description

Technical Field

[0001] The present invention relates to the technical field of biological sample detection, and in particular to a method for detecting urine steroid hormone metabolic profiles using solid phase extraction-ultra-performance liquid chromatography-tandem mass spectrometry. Background Art

[0002] Steroid hormones, also known as steroids or steroidal compounds, are a large class of cyclopentyl-fused perhydrophenanthrene derivatives synthesized from cholesterol in organs such as the adrenal glands, ovaries, testes, and adipose tissue. Steroid hormones participate in multiple physiological pathways in the body to maintain homeostasis, regulating the functions of cells, tissues, and organs throughout the life cycle. These include maintaining sexual differentiation and reproductive function, regulating lipid, glucose, and energy homeostasis, maintaining metabolic homeostasis, and regulating neuronal and immune function. The production and metabolism of steroid hormones involve the coordinated action of multiple enzyme systems, and their regulation is highly complex. Steroid hormone homeostasis is a key factor in maintaining normal physiological function in the human body.

[0003] The formation and transformation of steroid hormones and their metabolites are core links in maintaining endocrine balance. A single steroid hormone indicator can only reflect local endocrine imbalance and cannot capture the dynamic antagonistic or synergistic effects between hormones. The steroid hormone metabolic profile can systematically analyze the dynamic concentration changes of each hormone and its metabolites in the pathway. It can not only trace the metabolic sources of hormones, but also reveal the dynamic balance relationship between hormones through metabolic network analysis, which is helpful for the early prediction and diagnosis of complex endocrine diseases. In view of this, it is crucial to develop a detection method that can simultaneously quantify as many hormone substances as possible on the steroid hormone metabolic profile. Urine is an ideal biological sample for long-term monitoring studies because of its advantages such as non-invasiveness and easy and repeatable collection.

[0004] Currently, biological samples used for steroid hormone testing mainly include blood, saliva, urine, and tissue fluid. Although blood samples have a high degree of standardization, their collection is invasive and invasive, which limits their application in large-sample longitudinal studies of children. Although saliva samples can be collected non-invasively, the content of some steroid hormones is very low, which can easily lead to insufficient detection stability and increased variability in test results, making it difficult to meet the data stability requirements of metabolic profiling studies. The selection of biological samples requires a comprehensive consideration of the stability of the detection target, the convenience of sampling, and the representativeness of the metabolic characteristics. Among them, urine is suitable as a biological sample for this study because of its following significant advantages: 1) Urine is non-invasive and easy to collect repeatedly, making it more suitable as a biological sample for long-term monitoring studies in children; 2) The content of endogenous interfering substances in the urine matrix is ​​lower than that in other body fluids, which can obtain a better signal-to-noise ratio and improve the sensitivity and accuracy of mass spectrometry detection, which is particularly critical for low-concentration hormone detection in children; 3) Urine sample collection can effectively avoid hormone fluctuations caused by acute stress, ensuring that the sample can better reflect the basal metabolic state; 4) The urine metabolic profile of steroids provides a longer overview rather than just a point of insight, and allows for a more complete analysis of multiple metabolites at the same time.

[0005] For steroid hormone determination, immunoassays currently used in clinical laboratories offer advantages such as ease of operation and low cost. However, their technical limitations are hindering the development of precision medicine. These limitations primarily include: 1) antibody cross-reactivity, which can lead to false-positive / false-negative results; 2) difficulty distinguishing structurally similar steroid hormones (such as corticosterone (B) and cortisone (E)), which can easily lead to erroneous results; and 3) low sensitivity for detecting low-concentration steroid hormones (such as samples from children or patients with adrenal insufficiency), making these methods unsuitable for all clinical applications. Among instrumental analytical methods, the currently published gas chromatography-mass spectrometry (GC-MS) method requires derivatization, which is time-consuming and complex. Although liquid chromatography-mass spectrometry (LC-MS) has certain advantages in separation capabilities, most methods still rely on derivatization treatment to improve separation. Existing methods that do not require derivatization treatment have the limitation of only being able to detect a single or a few steroid hormones and cannot achieve a comprehensive analysis of the steroid hormone metabolic spectrum. Summary of the Invention

[0006] The purpose of the present invention is to address the above problems and provide a method for detecting the metabolic profile of urinary steroid hormones by solid phase extraction-ultra-performance liquid chromatography-tandem mass spectrometry.

[0007] In order to achieve its purpose, the present invention adopts the following technical solutions:

[0008] A method for detecting the metabolic profile of urinary steroid hormones by solid phase extraction-ultra-performance liquid chromatography-tandem mass spectrometry comprises the following steps:

[0009] S1. Prepare internal standard stock solution and target analyte standard stock solution

[0010] The target analyte is a urinary steroid hormone metabolic profile, and the urinary steroid hormone metabolic profile includes one or more of 17α-hydroxyprogesterone, progesterone, pregnenolone, corticosterone, 11-deoxycorticosterone, hydrocortisone, 11-deoxycortisol, cortisone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, 5α-dihydrotestosterone, androstenediol, androstenedione, androsterone, estradiol, and estriol;

[0011] S2. Pretreatment of urine samples to be tested

[0012] S2.1. Enzymatic hydrolysis: Take the urine to be tested, add the internal standard stock solution and the enzymatic hydrolysis solution, mix, and incubate to fully hydrolyze the uronic acid glycoside; the enzymatic hydrolysis solution contains glucuronidase;

[0013] S2.2, solid phase extraction separation and purification

[0014] a) Activating the solid phase extraction cartridge;

[0015] b) Sample loading: Loading the enzymatically treated urine sample onto a solid phase extraction cartridge;

[0016] c) elution: adding an eluent to wash the column, and then adding an acetonitrile / isopropanol mixture to elute the sample to obtain an eluent; the acetonitrile / isopropanol mixture is obtained by mixing the acetonitrile and isopropanol in a volume ratio of 5 to 7:3 to 5 (preferably 5.5 to 6.5:3.5 to 4.5);

[0017] S2.3. Multi-step solvent distribution and reconstitution

[0018] The collected eluate was blown dry, and an acetonitrile / dichloromethane mixture was added to redissolve the residue, centrifuged, and the supernatant was blown dry and redissolved with methanol to obtain a reconstituted solution; the acetonitrile / dichloromethane mixture was obtained by mixing acetonitrile and dichloromethane in a volume ratio of 2-5:8-5;

[0019] S2.4. Filter the reconstituted solution through a membrane filter;

[0020] S3. Urine sample analysis: The filtered reconstituted solution was subjected to high performance liquid chromatography-triple quadrupole tandem mass spectrometry;

[0021] The mass spectrometry was performed with an electrospray ionization source in the positive and negative ion modes using multiple reaction monitoring. The quantitative ion pairs in the mass spectrometry detection were: 17α-hydroxyprogesterone 303.0→109.0, progesterone 315.2→97.1, pregnenolone 299.0→159.3, corticosterone 361.2→163.1, 11-deoxycorticosterone 331.0→97.1, hydrocortisone 363.4→120.9, 11-deoxycortisol 347.2→1 09.1, cortisone 347.0→121.0, dehydroepiandrosterone 271.0→196.9, dehydroepiandrosterone sulfate 367.0→96.9, testosterone 289.4→96.9, 5α-dihydrotestosterone 291.0→255.0, androstenediol 273.0→255.6, androstenedione 287.0→97.1, androstenone 255.0→147.0, estradiol 255.0→159.0, estriol 289.0→107.0;

[0022] The mobile phase A in the liquid chromatography detection is 0.15-0.25% (V / V) acetic acid aqueous solution, and the mobile phase B is acetonitrile;

[0023] S4. Drawing of a standard curve: Using target analyte standard stock solutions, ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry was performed to determine the linear relationship between the peak area ratio of the analyte relative to the internal standard minus the peak area ratio of the analyte relative to the internal standard in the blank sample (y) and the concentration of each analyte (x), to obtain a standard curve for the target analyte;

[0024] S5. Data analysis: The analyte was quantified by the internal standard method based on the ratio of the target peak area to the internal standard peak area.

[0025] Preferably, in step S2.2, a) activating the solid phase extraction cartridge: sequentially activating the Oasis HLB cartridge with methanol and water, preferably sequentially activating the Oasis HLB cartridge with 1.8-2.5 mL of methanol and 1.8-2.5 mL of water, wherein the specification of the Oasis HLB cartridge is 200 mg / 6 mL.

[0026] Preferably, in step S2.2, the eluent is a 4-6% (V / V) (preferably 4.5-5.5%) methanol solution, the volume of the eluent is preferably 2-7 mL (further preferably 4-6 mL or 5-6 mL), and the volume of the acetonitrile / isopropanol mixture is 2-4 mL (preferably 2-3 mL).

[0027] Preferably, step S2.2 is performed using a high-throughput fully automatic solid phase extraction instrument, and the SPE operation is performed at a fixed flow rate of 1 mL / min, and standardized air push is performed between each step: after each step is completed and before the next step begins, air push is performed at a speed of 10 mL / min for 0.6 min to ensure that no liquid remains on the solid phase extraction column.

[0028] Preferably, in step S2.4, an organic nylon filter provided with a 0.22 μm filter membrane is used for filtration.

[0029] Preferably, the acetonitrile / isopropanol mixture in step S2.2 is obtained by mixing acetonitrile and isopropanol in a volume ratio of 6:4;

[0030] The acetonitrile / dichloromethane mixed solution in step S2.3 is obtained by mixing acetonitrile and dichloromethane in a volume ratio of 2.5-3.5:6.5-7.5 (preferably 3:7).

[0031] Preferably, the internal standard stock solution and the target analyte standard stock solution are prepared using methanol; the internal standard is methyltestosterone, and the quantitative ion pair in the mass spectrometry detection of methyltestosterone is 313.0→271.0.

[0032] Preferably, in step S2.1, the enzymatic hydrolysis application solution contains β-glucuronidase at a concentration of 500 to 2500 units / mL (preferably 1000 to 2000 units / mL) and sulfatase at a concentration of 250 to 550 units / mL (preferably 350 to 450 units / mL); it is preferably prepared using an ammonium acetate-glacial acetic acid solution with a pH of 5 to 6.

[0033] Preferably, in step S2.1, the incubation is culturing at 35-37° C. for 8-20 hours (preferably 10-20 hours or 15-16 hours);

[0034] The ammonium acetate-glacial acetic acid buffer solution has an ammonium acetate concentration of 0.8 to 1.2 mmol / mL and is adjusted to a pH of 5 to 6 (preferably pH 5.5) using glacial acetic acid.

[0035] Preferably, in step S3, the liquid chromatography separation column is ACQUITY BEH C18, 2.1 mm × 150 mm, 1.7 mm particle size, column temperature 35 ° C ~ 45 ° C (preferably 40 ° C); injection volume 4 to 10 μL (preferably 5 μL); elution conditions are gradient elution: flow rate 0.2 to 0.5 mL / min (preferably 0.30 mL / min), conditions are: 0 min, 80% to 90% A (preferably 80%); 4 to 6 min (preferably 5 min), 75% to 80% A (preferably 75%) ); 6-9 min (preferably 8 min), 70%-75% A (preferably 70%); 9-12 min (preferably 10 min), 65%-70% A (preferably 65%); 12-15 min (preferably 14 min), 25-35% A (preferably 30% A), 15 min-17 min (preferably 16 min), 75-85% A (preferably 80% A), hold for 2-4 min (preferably 2 min).

[0036] The beneficial effects of the present invention are:

[0037] The polarity differences of multi-step solvents are used to achieve the selective separation of impurities of different properties in steps. High-polarity components are preferentially washed by low-polarity solvents, and medium-polarity impurities are gradient eluted by mixed solvents. Finally, the target product is recovered in a strong polarity solvent. This process significantly improves the purification efficiency while avoiding the loss of key analytes. Compared with the urine pretreatment methods currently reported in the literature, this experiment significantly reduces the matrix effect of urine. In addition, the SPE sample pretreatment operation selected in this study is accurately completed with the help of a fully automatic solid phase extraction instrument, avoiding the decrease in column efficiency or loss of target products due to uneven flow rate during manual operation, eliminating the interval delay in manual operation to a certain extent, reducing the accidental errors and labor costs caused by differences in operator habits, and effectively improving experimental efficiency. By optimizing the enzymatic hydrolysis conditions, elution conditions, chromatography, and mass spectrometry, the minimum detection limit for different analytes ranged from 0.001 to 0.027 ng / mL, the minimum quantification limit ranged from 0.005 to 0.089 ng / mL, and the average recovery of the target compounds at three different spiked concentrations was 98.58%–100.92%. The intra-day and inter-day precision were tested, and the method had satisfactory reproducibility for all analytes (RSD < 3.01%).

[0038] Compared with the existing technology, the method of the present invention has lower detection limits and quantification limits and higher sensitivity. Moreover, due to the lower detection limits and quantification limits, the method of the present invention can be applied to the detection of special populations, such as patients with adrenal insufficiency, gonadal dysgenesis, patients with congenital enzyme deficiency, etc., and has a wider range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The effect of different enzymatic digestion times on the mass spectrometry signal response of the target compound is shown.

[0040] Figure 2 The effect of different enzyme amounts on the mass spectrometry signal response of the target compound is shown.

[0041] Figure 3 This is a graph showing the percentage ratio of free steroid hormones to bound steroid hormones in urine.

[0042] Figure 4 The effect of different eluents on the MS signal response of the target compound is shown.

[0043] Figure 5 The effect of different solid phase extraction columns on the MS signal response of the target compounds is shown.

[0044] Figure 6 The effect of different eluent volumes on the MS signal response of the target compound is shown.

[0045] Figure 7 The effect of different types of eluents on the MS signal response of the target compound is shown.

[0046] Figure 8 The effect of different eluent volumes on the MS signal response of the target compound is shown.

[0047] Figure 9 The effects of different types of reconstitution solutions on the MS signal response of the target compound are shown.

[0048] Figure 10 The effects of three different dilution schemes on the MS signal response of the target compound are shown.

[0049] Figure 11 This is the total ion current of steroid hormones in urine measured by HPLC-MS / MS.

[0050] Figure 12 The effect of different mobile phase combinations on the MS signal response of the target compound. DETAILED DESCRIPTION

[0051] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0052] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0053] Example 1

[0054] 1 Experimental instruments and materials

[0055] The experimental instruments and materials used in the present invention are shown in Table 1.

[0056] Table 1 Experimental instruments and materials

[0057]

[0058]

[0059] 2 Standards and experimental reagents

[0060] The standard substances and experimental reagents used in the present invention are shown in Table 2 and Table 3.

[0061] Table 2 Standards

[0062]

[0063] Table 3 Experimental reagents

[0064]

[0065] The urine sample to be tested comes from: urine sample of student volunteers from a primary school in Chongqing.

[0066] 3. Preparation of standard solutions and reagents

[0067] Stock solutions: Weigh appropriate amounts of steroid hormone standards and methyltestosterone internal standard (IS) solids into 2 mL brown tubes and dissolve in methanol to prepare single-standard stock solutions at an accurate concentration of 1 mg / mL. Next, pipette 60 μL of each of the 17 single-standard steroid hormone stock solutions into 980 μL of methanol in a 5 mL polypropylene (PP) tube to prepare a 30 μg / mL mixed standard stock solution. Mix thoroughly and transfer to a brown glass bottle. Store these stock solutions at -20°C until use.

[0068] Preparation of the enzymatic hydrolysis solution: Add 120 μL of the β-glucuronidase / arylsulfatase liquid enzyme solution to 3 mL of a pH 5.5 ammonium acetate-glacial acetic acid solution to prepare an enzymatic hydrolysis solution containing 2000 units / mL of β-glucuronidase and 400 units / mL of sulfatase. Shake thoroughly and use immediately. To prepare the ammonium acetate-glacial acetic acid solution: Accurately weigh 1.541 g of ammonium acetate and immerse it in 20 mL of pure water (i.e., an ammonium acetate concentration of 1 mmol / mL). Adjust the pH to 5.5 with glacial acetic acid.

[0069] 4 Experimental methods

[0070] 4.1 Urine sample pretreatment

[0071] ① Enzymatic hydrolysis: Add 2 mL of urine sample, 10 μL of 30 μg / mL methyltestosterone internal standard (IS), and 500 μL of enzymatic hydrolysis solution to a 50 mL centrifuge tube and incubate overnight at 36.5°C in a constant temperature incubator for 16 h to fully hydrolyze the uronic acid glycoside.

[0072] ②Solid phase extraction combined with multi-step solvent distribution purification:

[0073] a) Automation: The extraction was performed precisely on a high-throughput, fully automated solid-phase extraction instrument at a flow rate of 1 mL / min. After each step and before the next, air was pushed at a rate of 10 mL / min for 0.6 min to ensure that no liquid remained on the solid-phase extraction column.

[0074] b) Column activation: Activate an Oasis HLB (200 mg / 6 mL) column with 2 mL of methanol and then 2 mL of water;

[0075] c) Sample loading: 2 mL of urine sample that had been enzymatically treated (37°C, 2 h) and vortex-mixed (1000 rpm, 5 min) was loaded onto the column;

[0076] d) Elution: Add 6 mL of 5% methanol to wash the column, and then add 4 mL of acetonitrile / isopropanol (6:4, v / v) to elute the sample.

[0077] ③ Multi-step solvent partitioning and reconstitution: Evaporate the collected eluate to dryness under nitrogen at 45°C, then add 1 mL of acetonitrile / dichloromethane (3:7, v / v) to reconstitute the residue. Centrifuge at 5000 rpm for 5 min at 4°C. Transfer the supernatant to a fresh PP tube and evaporate to dryness under nitrogen at 45°C. Dissolve the residue in 300 μL of methanol and transfer it to a 1 mL sterile syringe.

[0078] ④ Filtration: Inject the liquid in the sterile syringe into a 0.22 μm organic nylon filter and filter it into a 1.5 mL brown injection bottle equipped with a 300 μL inner liner for HPLC-MS / MS analysis.

[0079] 4.2 Urine sample testing

[0080] Using SCIEX QTRAP TM The analysis was performed on a 6500+ HPLC-MS / MS system. The electrospray ionization source operated in positive ion mode (ESI+) and negative ion mode (ESI-) by multiple reaction monitoring (MRM). The quantitative ion pairs in mass spectrometry detection were 17α-hydroxyprogesterone 303.0→109.0, progesterone 315.2→97.1, pregnenolone 299.0→159.3, corticosterone 361.2→163.1, 11-deoxycorticosterone / 21-hydroxyprogesterone 331.0→97.1, cortisol / hydrocortisone 363.4→120.9, 11-deoxycortisol 347.2→109.1, cortisone 347.0→121.0, Dehydroepiandrosterone 271.0→196.9, dehydroepiandrosterone sulfate 367.0→96.9, testosterone 289.4→96.9, 5α-dihydrotestosterone 291.0→255.0, androstenediol 273.0→255.6, androstenedione 287.0→97.1, androstenone 255.0→147.0, estradiol 255.0→159.0, estriol 289.0→107.0, methyltestosterone 313.0→271.0.

[0081] Chromatographic separation was performed on an ACQUITY BEH C18 column (2.1 mm × 150 mm, 1.7 mm particle size, Waters, Milford, MA, USA). The injection volume was 5 μL, and the column was maintained at 40°C. The mobile phase consisted of 0.2% acetic acid in water (A) and acetonitrile (B). Gradient elution was performed at a flow rate of 0.30 mL / min using the following conditions: 0 min, 80% A; 5 min, 75% A; 8 min, 70% A; 10 min, 65% A; 14 min, 30% A; 16 min, 80% A, and a 2-min hold.

[0082] 4.3 Standard curve drawing

[0083] The mixed standard stock solution was serially diluted with methanol to prepare mixed standard application solutions at concentrations of 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, and 2000 ng / mL. A 13-point calibration curve ranging from 0 to 2000 ng / mL was constructed. The calibration curve was subjected to least-squares linear regression in triplicate to determine the linear relationship of the method.

[0084] 5 Results

[0085] 5.1 Optimization of sample pretreatment conditions

[0086] 5.1.1 Optimization of enzymatic hydrolysis conditions

[0087] 1. Optimization of enzymatic hydrolysis time

[0088] This experiment was carried out under the conditions of fixed enzyme amount (containing 2000 units / mL of glucuronidase and 400 units / mL of sulfatase), fixed pH (pH=5.5) and fixed incubation temperature (36.5℃). The enzymatic hydrolysis time gradient was set to 1h, 2h, 3h, 4h, 15h, and 16h. Three parallel samples were set up for each condition. The final enzymatic hydrolysis time was determined according to the mass spectrometry signal response value of the steroid hormone. The experimental results are shown in ( Figure 1 ) showed that, except for E3, which had similar response values ​​at different enzymatic incubation times, the peak areas of the other target compounds were all maximized when the enzymatic incubation time was 16 h. Therefore, 16 h was selected as the enzymatic incubation time for this experiment.

[0089] 2. Enzyme dosage optimization

[0090] Based on the optimal enzymatic hydrolysis time (16 h) determined above, under the conditions of fixed incubation temperature and pH of the enzymatic hydrolysis solution, the enzyme amount gradient was set to 500 units / mL of glucuronidase / 100 units / mL of sulfatase, 1000 units / mL of glucuronidase / 200 units / mL of sulfatase, and 2000 units / mL of glucuronidase / 400 units / mL of sulfatase to perform the experimental operation. Three parallel samples were set up for each condition, and the final enzymatic hydrolysis time was determined based on the mass spectrometry signal response value of the steroid hormone. The experimental results are shown in ( Figure 2 ) showed that the peak area of ​​the target compound continued to increase with increasing enzyme dosage. Therefore, 2000 units / mL of glucuronidase and 400 units / mL of sulfatase were selected as the enzyme dosages for the enzymatic hydrolysis in this experiment.

[0091] 3. Comparison between enzymatic hydrolysis and non-enzymatic hydrolysis

[0092] Most steroid hormones are fat-soluble molecules, which will bind to polar molecules such as glucuronic acid or sulfuric acid in urine to enhance their water solubility. This experiment compared the peak area of ​​the analytes in urine samples with and without enzymatic hydrolysis. The enzymatic hydrolysis of urine samples used the optimal enzymatic hydrolysis conditions determined by the above experiment. The same volume of diluent (methanol) was used to replace the enzyme solution for the unenzymatically hydrolyzed samples. Other conditions were consistent with the enzymatic hydrolysis sample processing operation. The peak area detected after enzymatic hydrolysis was used as the total peak area, and the percentage of free steroid hormones and bound steroid hormones in urine was calculated. Experimental results ( Figure 3 ) showed that all 17 hormones in the urinary steroid hormone metabolic profile tested in this study existed in bound form, with the proportion of bound form ranging from 16% to 79%, suggesting the necessity of enzymatic hydrolysis of urine before measurement.

[0093] 5.1.2 Selection of solid phase extraction columns

[0094] Three solid phase extraction cartridges were compared: Strata@C18-E (55μm, 70A) 100mg / 1mL, Tubes, 100 / pk [Phenomenex C18-E]; Oasis HLB 6cc Vac Cartridge, 200mg Sorbent per Cartridge, 30μm, 30 / pk [Oasis HLB]; and Styra HLB 6cc Vac Cartridge, 200mg Sorbent per Cartridge, 50μm, 30 / pk [SHIMSEN].

[0095] Phenomenex C 18 -E column experimental results ( Figure 5A) shows that although the peak areas of preg and 5-diol are relatively high when 90% methanol is used as the elution reagent, the peak areas are not much different from those when 100% methanol is used as the elution reagent. It is worth noting that the peak area of ​​4-dione is much higher when 100% methanol is used as the elution reagent than when 90% methanol is used as the elution reagent. Therefore, considering all factors, it was decided to use Phenomenex C 18 -E column, 100% methanol is the optimal elution reagent.

[0096] Experimental results of Oasis HLB column ( Figure 5 Figure B) shows that most target compounds have higher peak areas when eluting with 90% methanol, with 4-dione in particular showing a much higher peak area than under the other two conditions. While prog, DHT, and 5-diol do not have the highest peak areas when eluting with 90% methanol, the responses of the less responsive preg and B are significantly lower when eluting with 100% methanol. Similarly, the responses of the less responsive E3, AN, and prog are significantly lower when eluting with 80% methanol. Therefore, after comprehensive consideration, 90% methanol was determined to be the optimal elution reagent for Oasis HLB columns.

[0097] Experimental results of SHIMSEN HLB column ( Figure 5 C) shows that, with the exception of slightly decreased peak areas for DHEA, 17α-Hydroxydine, and 4-dione, the peak areas of most target compounds were higher when eluted with 100% methanol. Therefore, considering all factors, 100% methanol was determined to be the optimal elution agent for Shimsen HLB columns.

[0098] Next, based on the above experimental results, the elution efficiency under three conditions, namely Phenomenex C18-E (100% methanol), Oasis HLB (90% methanol), and SHIMSEN HLB (100% methanol), was further compared, with three parallel samples set up for each condition. The experiment found that the sample treated with the Oasis HLB column was more transparent and clearer than the sample solution treated with the other two columns under naked eye observation, and the mass spectrometry results showed that its baseline was lower, and the peak areas of the target substances of most steroid hormones (B, DHEA, F, 17α-Hyd, E, DHT, S, 21-Hydr, 4-dione) were significantly higher than the peak areas under other conditions, indicating that the column can better retain the target substances while removing more impurities. Although the results show that the peak areas of 5-diol, DHEAS, and AN are higher in the samples treated with the SHIMSEN HLB column, there is almost no response to preg with a low response value, so Phenomenex C 18The -E column is not suitable for comprehensive analysis. Finally, after comprehensive consideration, the Oasis HLB column was selected for the extraction and purification of steroid hormones in urine.

[0099] 5.1.3 Optimization of SPE Combined with Multi-step Solvent Distribution

[0100] 5.1.3.1 Optimization of elution procedures

[0101] 1. Optimization of eluent types

[0102] The following five groups of water-methanol mixed solutions with different ratios were used as eluents to investigate the removal of interfering substances in urine matrix. Three parallel samples were set up for each condition. (1) H2O (2) 5% methanol (3) methanol: 0.1% acetic acid water (3:7, v / v) (4) 45% methanol (5) 5% methanol: 40% methanol (2:1, v / v). The experimental results are shown in the figure. Figure 4 The results show that 5% methanol as the eluent effectively removes impurities from the sample while retaining the target compounds, as evidenced by the higher peak areas of most targets under this eluent. It is worth noting that although the peak areas of 17α-Hyd, B, E, S, and F are slightly lower under this eluent than under other wash conditions, it significantly improves the response of low-response hormones (E3, prog), leading to the selection of 5% methanol as the eluent for this experiment.

[0103] 2. Eluent volume optimization

[0104] This study investigated the effects of five eluent volumes of 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL on sample treatment. The results are shown in Figure 6 We can observe that as the volume of the eluent increases, the urine sample becomes clearer and more transparent. The experimental results also show that the baseline values ​​of the samples are generally smaller, and the peak areas of the target substances are generally larger. Finally, 6 mL was selected as the eluent volume for this experiment.

[0105] 5.1.3.2 Elution program optimization

[0106] 1. Optimization of eluent types

[0107] The study compared the elution efficiency of the target steroid hormones using the following six eluents, with three parallel samples set up for each condition. (1) Methanol: dichloromethane = 1:1 (v / v) (2) Methanol: water = 8:2 (v / v) (3) Acetonitrile: isopropanol = 6:4 (v / v) (4) Methanol: water = 9:1 (v / v) (5) Acetonitrile: dichloromethane = 3:7 (v / v) (6) Acetonitrile: dichloromethane = 1:1 (v / v), ethyl acetate: dichloromethane = 1:1 (v / v), methanol: chloromethane = 1:1 (v / v), and passed through the column in sequence. Experimental results ( Figure 7) showed that the elution efficiency was relatively high when acetonitrile:isopropanol = 6:4 (v / v) was used as the eluent of this method, which was reflected in the fact that the peak areas of most target compounds were larger than those under other conditions. Finally, acetonitrile:isopropanol = 6:4 (v / v) was selected as the eluent for this experiment.

[0108] 2. Optimization of eluent volume

[0109] The elution effects of five elution volumes, 2mL, 3mL, 4mL, 5mL, and 6mL, on the target analytes were investigated. Figure 8 ) showed that increasing the elution volume from 2 mL to 4 mL increased the peak areas of most targets. However, increasing the volume above 4 mL showed a decreasing trend in the peak areas of the less sensitive preg and E3. Therefore, after comprehensive considerations, 4 mL was selected as the eluent volume for this experiment.

[0110] 5.1.3.3 Optimization of the first concentration and reconstitution procedure

[0111] The sample still had a slightly yellowish color after elution, indicating that some impurities (such as pigments and medium-polarity organic matter) remained in the urine and had not been completely removed. Therefore, we added 1 mL of acetonitrile-dichloromethane (3:7, v / v) as a concentrated reconstitution reagent to purify the sample again. The low polarity of dichloromethane and the medium polarity of acetonitrile synergistically enabled the target steroid hormone to be efficiently dissolved. Impurities such as medium-polarity organic matter, residual lipids, and other non-target metabolites had their solubility reduced due to the change in the polarity gradient of the mixed solvent, resulting in selective precipitation.

[0112] 5.1.3.4 Optimization of the Second Concentration and Redissolution Procedure

[0113] We investigated which of five different reagents (methanol, 50% methanol, 50% acetonitrile, 0.2% acetic acid water:acetonitrile = 8:2 (v:v), water:methanol:acetonitrile = 8:1:1 (v:v:v)) could best dissolve the target steroid hormone when used as a reconstitution solution. The experimental results showed that ( Figure 9 ). When methanol was used as the reconstitution solution, although the responses for DHEA and 4-dione, two steroid hormones with inherently high responses, were not the highest, considering that E3, which has the lowest sensitivity, had the highest response under this reconstitution condition, we ultimately chose methanol as the reconstitution solution for this experiment after comprehensive consideration.

[0114] 5.1.4 Optimization of dilution conditions for steroid hormone standards

[0115] We investigated three different dilution schemes, as follows:

[0116] Solution 1: Dilute all standards with methanol to prepare mixed standard intermediate solution 1 (10 μg / mL);

[0117] Solution 2: All standards were diluted with the initial mobile phase (0.2% acetic acid in water: acetonitrile = 8:2, v / v) to form mixed standard intermediate solution 2 (10 μg / mL);

[0118] Solution 3: Dilute 7 steroid hormone standards with the initial mobile phase to form mixed standard intermediate solution 3 (10 μg / mL), and dilute the remaining 11 steroid hormone standards with methanol to form mixed standard intermediate solution 4 (10 μg / mL). The experimental results show that ( Figure 10 ): When operating according to Scheme 1, the response values ​​of most target analytes were relatively the highest. Although the response value of 5-diol was slightly lower than that of other schemes, the response values ​​of several steroid hormones in other schemes were too low (preg, E3, 17α-Hy d, F, E, etc.). Based on comprehensive considerations, Scheme 1 was selected as the dilution operation for the standard in this experiment.

[0119] 5.2 Optimization of mass spectrometry conditions

[0120] In this experiment, a SCIEX QTRAP 6500+ HPLC-MS / MS instrument was used in MRM mode. Parameters such as ion pairs, cone voltage, and collision voltage were optimized to ensure good mass spectrometric response signals for both parent and product ions of 17 steroid hormones. The final optimized mass spectrometric parameters are shown in Table 4.

[0121] Table 4

[0122]

[0123]

[0124] 5.3 Optimization of chromatographic conditions

[0125] This study used a binary mobile phase, with component A being an aqueous phase and component B being an organic phase. The effects of four different mobile phase combinations on the separation of steroid hormones were investigated: (1) A—pure water, B—acetonitrile; (2) A—0.2% (V / V) acetic acid aqueous solution, B—acetonitrile; (3) A—pure water, B—methanol; (4) A—0.2% acetic acid aqueous solution, B—methanol. The effects of different mobile phase combinations on the MS signal response of the target compounds are shown in Figure 2. Figure 12 The results show that when "A-0.2% acetic acid aqueous solution, B-acetonitrile" is used as the mobile phase, the separation effect is the best and the peak shape is better. The chromatogram is shown in Figure 11 .

[0126] 5.4 Methodological evaluation

[0127] 5.4.1 Linear range, minimum detection limit, minimum quantification limit, and matrix effect

[0128] We tested the linear relationship between the peak area ratio of the analyte to the internal standard in the spiked sample minus the peak area ratio of the analyte to the internal standard in the blank sample (y) and the concentration of each analyte (x). The results, listed in Table 5, demonstrate excellent linearity. The minimum detection limit ranged from 0.001 to 0.027 ng / mL, and the minimum quantification limit ranged from 0.005 to 0.089 ng / mL for the various analytes. Matrix effects for the 17 target compounds ranged from -8.7% to 8.5%.

[0129] Table 5 Methodological validation parameters for target analytes in urine: linearity, limit of quantification (LOQ), limit of detection (LOD), and matrix effect (ME%)

[0130]

[0131]

[0132] 5.4.2 Recovery and precision

[0133] A systematic evaluation of the recoveries and precision of 17 steroid hormones in urine was conducted at low, medium, and high spike concentration levels. The results are shown in Table 6. The average recoveries of all target compounds at different spike concentrations ranged from 98.58% to 100.92%.

[0134] The precision results showed satisfactory reproducibility, with intra-day RSDs of all targets ≤3.01% and inter-day RSDs of all targets ≤1.83%.

[0135] Table 6 Recovery (%), intra-day and inter-day precision (RSD, %) of target compounds in urine at three spiked concentrations

[0136]

[0137]

[0138] 5.4.3 Stability

[0139] As shown in Table 7, the target compound loss rate was less than 5% after the urine sample extract was stored in the dark at room temperature (25°C) for 24 hours and refrigerated (4°C) for 3 days. Furthermore, after three freeze-thaw cycles (-20°C), the target compound remained stable, with a loss rate of no more than 3.8%, which was not significantly different from the non-freeze-thawed sample.

[0140] Table 7 Stability of target compounds in urine under different operating conditions

[0141]

[0142] 5.4.4 Comparison of detection methods

[0143] The performance indicators of the method of the present invention were compared with those of the prior art steroid hormone detection methods. The comparison results are shown in Table 8.

[0144] Table 8 Comparison of performance indicators of different steroid hormone detection methods

[0145]

[0146] In Table 8, the documents compared with the present invention are as follows:

[0147] Document 1: Dai W, Huang Q, YinP, et al. Comprehensive and highly sensitive steroid hormone profiling method based on stable isotope-labelingliquid chromatography-mass spectrometry[J]. Analytical Chemistry, 2012, 84(23): 10245-51.

[0148] Document 2: Li ZM, Kannan K. Determination of 19 Steroid Hormones in HumanSerum and Urine Using Liquid Chromatography-Tandem Mass Spectrometry[J]. Toxics, 2022,10(11):687. Document 3: Zhang X, Yang N, Wang M, et al. Establishment and validation of a sensitive LC-MS / MS method for quantification of urinaryestrogens in women with gestational diabetes mellitus[J].Journal ofPharmaceutical and Biomedical Analysis,2022,221:115044.

[0149] File 4: Struck-Lewicka W, Karpińska B, Rodzaj W, et al. Development of thethin film solid phase microextraction (TF-SPME) method for metabolomics profiling of steroidal hormones from urine samples using LC-QTOF / MS[J]. Frontiers in Molecular Biosciences, 2023,10:1074263.

[0150] Document 5: Dong H, Hu B, Chen W, et al. ALC-MS / MS method for the simultaneous quantitative determination of aldosterone, its precursor 18-hydroxycorticosterone and its metabolite tetrahydroaldosterone in human urine [J]. Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences, 2024, 1234: 124012.

[0151] High performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS / MS) has the characteristics of high specificity, high sensitivity and multi-component simultaneous detection capabilities, and can become a key analytical means to break through the bottleneck of traditional methods. HPLC-MS / MS technology can effectively distinguish structural analogs through accurate mass screening of the quadrupole mass analyzer, combined with characteristic fragment ion comparison; at the same time, based on the dynamic multiple reaction monitoring (MRM) mode, it can also achieve an ultra-low detection limit level of picomolar level, which is particularly suitable for the accurate quantitative analysis of trace steroid hormones in complex matrices (such as serum and urine). The present invention is based on HPLC-MS / MS, and the study explored the influence of different experimental conditions on the detection results, and finally determined a detection method with high precision, high result accuracy, good stability, high sensitivity, good reproducibility, and the ability to simultaneously quantify as many hormone substances as possible on the steroid hormone metabolic spectrum:

[0152] 1. Improved multi-step solvent partitioning purification process: Using an acetonitrile-isopropanol (6:4, v / v) mixed solvent as the eluent significantly improved the recovery rate of 17 target steroid hormones. The innovative introduction of an acetonitrile-dichloromethane (3:7, v / v) reconstitution step utilizes polarity differences to achieve selective separation of impurities. Final reconstitution in methanol improves detection sensitivity.

[0153] 2. Optimization of fully automated solid-phase extraction technology: The entire process was precisely operated on a high-throughput fully automated solid-phase extraction instrument, with a fixed flow rate of 1 mL / min for SPE operation and standardized gas push (10 mL / min, 0.6 min) between each step to avoid accidental errors caused by manual operation and significantly improve experimental efficiency and reproducibility.

[0154] 3. Optimization of the combination of Oasis HLB solid-phase extraction cartridges and elution conditions: A specific specification of Oasis HLB cartridge (200 mg / 6 mL) was selected; the eluent was optimized to 6 mL of 5% methanol solution to effectively remove matrix interference; and the eluent volume was optimized to 4 mL.

[0155] 4. Systematic optimization of enzymatic hydrolysis conditions: The dosage of β-glucuronidase was optimized to 2000 units / mL, and that of sulfatase to 400 units / mL; the enzymatic hydrolysis time was extended to 16 hours to ensure sufficient hydrolysis of the bound hormone; and the enzymatic hydrolysis was performed in ammonium acetate-glacial acetic acid buffer at pH 5.5 to enhance enzyme activity.

[0156] 5. Specific chromatography-mass spectrometry conditions: Column: ACQUITY BEH C18 (2.1 mm × 150 mm, 1.7 mm particle size); Mobile phase: Optimized gradient elution program of 0.2% acetic acid aqueous solution (A)-acetonitrile (B); Specific MRM ion pair parameter settings enable high-sensitivity detection of 17 steroid hormones.

[0157] The present invention utilizes the polarity difference of multi-step solvents to realize the selective separation of impurities of different properties step by step. High polarity components are preferentially washed by low polarity solvents, medium polarity impurities are eluted by mixed solvent gradient, and the final target is recovered in a strong polar solvent. This process significantly improves the purification efficiency while avoiding the loss of key analytes. Compared with the urine pretreatment method reported in the current literature, this experiment significantly reduces the matrix effect of urine. In addition, the SPE sample pretreatment operation selected by the present invention is accurately completed by means of a fully automatic solid phase extractor, avoiding the column efficiency drop or target loss caused by uneven flow rate in manual operation, eliminating the interval delay in manual operation to a certain extent, reducing the accidental error and labor cost caused by operator habit differences, and effectively improving the experimental efficiency. By optimizing the enzymatic hydrolysis conditions, elution conditions, chromatography, and mass spectrometry, the minimum detection limit for different analytes ranged from 0.001 to 0.027 ng / mL, the minimum quantification limit ranged from 0.005 to 0.089 ng / mL, and the average recovery of the target compounds at three different spiked concentrations was 98.58%–100.92%. The intra-day and inter-day precision were tested, and the method had satisfactory reproducibility for all analytes (RSD < 3.01%).

Claims

1. A method for detecting the metabolic profile of urinary steroid hormones by solid phase extraction-ultra-performance liquid chromatography-tandem mass spectrometry, characterized in that: The steps include: S1. Prepare internal standard stock solution and target analyte standard stock solution The target analyte is a urinary steroid hormone metabolic profile, and the urinary steroid hormone metabolic profile includes one or more of 17α-hydroxyprogesterone, progesterone, pregnenolone, corticosterone, 11-deoxycorticosterone, hydrocortisone, 11-deoxycortisol, cortisone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, 5α-dihydrotestosterone, androstenediol, androstenedione, androsterone, estradiol, and estriol; S2. Pretreatment of urine samples to be tested S2.

1. Enzymatic hydrolysis: Take the urine to be tested, add the internal standard stock solution and the enzymatic hydrolysis solution, mix, and incubate to fully hydrolyze the uronic acid glycoside; the enzymatic hydrolysis solution contains glucuronidase; S2.2, solid phase extraction separation and purification a) Activating the solid phase extraction cartridge; b) Sample loading: Loading the enzymatically treated urine sample onto a solid phase extraction cartridge; c) elution: adding an eluent to wash the column, and then adding an acetonitrile / isopropanol mixture to elute the sample to obtain an eluent; the acetonitrile / isopropanol mixture is obtained by mixing the acetonitrile and isopropanol in a volume ratio of 5 to 7:3 to 5; S2.

3. Multi-step solvent distribution and reconstitution The collected eluate was blown dry, and an acetonitrile / dichloromethane mixture was added to redissolve the residue, centrifuged, and the supernatant was blown dry and redissolved with methanol to obtain a reconstituted solution; the acetonitrile / dichloromethane mixture was obtained by mixing acetonitrile and dichloromethane in a volume ratio of 2-5:8-5; S2.

4. Filter the reconstituted solution through a membrane filter; S3. Urine sample analysis: The filtered reconstituted solution was subjected to high performance liquid chromatography-triple quadrupole tandem mass spectrometry; The mass spectrometry was performed with an electrospray ionization source in the positive and negative ion modes using multiple reaction monitoring. The quantitative ion pairs in the mass spectrometry detection were: 17α-hydroxyprogesterone 303.0→109.0, progesterone 315.2→97.1, pregnenolone 299.0→159.3, corticosterone 361.2→163.1, 11-deoxycorticosterone 331.0→97.1, hydrocortisone 363.4→120.9, 11-deoxycortisol 347.2→1 09.1, cortisone 347.0→121.0, dehydroepiandrosterone 271.0→196.9, dehydroepiandrosterone sulfate 367.0→96.9, testosterone 289.4→96.9, 5α-dihydrotestosterone 291.0→255.0, androstenediol 273.0→255.6, androstenedione 287.0→97.1, androstenone 255.0→147.0, estradiol 255.0→159.0, estriol 289.0→107.0; The mobile phase A in the liquid chromatography detection is 0.15-0.25% (V / V) acetic acid aqueous solution, and the mobile phase B is acetonitrile; S4. Drawing of a standard curve: Using target analyte standard stock solutions, ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry was performed to determine the linear relationship between the peak area ratio of the analyte relative to the internal standard minus the peak area ratio of the analyte relative to the internal standard in the blank sample (y) and the concentration of each analyte (x), to obtain a standard curve for the target analyte; S5. Data analysis: The analyte was quantified by the internal standard method based on the ratio of the target peak area to the internal standard peak area.

2. The method according to claim 1, wherein: In step S2.2, a) activating the solid phase extraction cartridge: sequentially activating the Oasis HLB cartridge with methanol and water, preferably sequentially activating the Oasis HLB cartridge with 1.8-2.5 mL of methanol and 1.8-2.5 mL of water, wherein the specification of the Oasis HLB cartridge is 200 mg / 6 mL.

3. The method according to claim 1, wherein: In step S2.2, the eluent is a 4-6% (V / V) methanol solution, preferably the volume of the eluent is 2-7 mL, and the volume of the acetonitrile / isopropanol mixture is 2-4 mL.

4. The method according to claim 1, wherein: Step S2.2 was performed using a high-throughput fully automatic solid-phase extraction instrument with a fixed flow rate of 1 mL / min for SPE operation. Standardized air push was performed between each step: air push was performed at a rate of 10 mL / min for 0.6 min after each step and before the next step to ensure that no liquid remained on the solid-phase extraction column.

5. The method according to claim 1, wherein: In step S2.4, filtration is performed using an organic nylon filter equipped with a 0.22 μm filter membrane.

6. The method according to claim 1, wherein: The acetonitrile / isopropanol mixture described in step S2.2 is mixed according to a volume ratio of acetonitrile to isopropanol of 6:4; The acetonitrile / dichloromethane mixed solution in step S2.3 is obtained by mixing acetonitrile and dichloromethane in a volume ratio of 2.5-3.5:6.5-7.

5.

7. The method according to claim 1, wherein: The internal standard stock solution and the target analyte standard stock solution were prepared using methanol; the internal standard was methyltestosterone, and the quantitative ion pair in the mass spectrometry detection of methyltestosterone was 313.0→271.

0.

8. The method according to claim 1, wherein: In step S2.1, The enzymatic hydrolysis application liquid contains 500-2500 units / mL of beta-glucuronidase and 250-550 units / mL of sulfatase; and is preferably prepared by using an ammonium acetate-glacial acetic acid solution with a pH of 5-6.

9. The method according to claim 8, characterized in that: In step S2.1, the incubation is performed at 35-37° C. for 8-20 hours; The ammonium acetate-glacial acetic acid buffer solution has an ammonium acetate concentration of 0.8 to 1.2 mmol / mL and is adjusted to a pH of 5 to 6 using glacial acetic acid.

10. The method according to claim 1, wherein: In step S3, the chromatographic column selected for liquid chromatography separation is ACQUITYBEH C18, 2.1 mm × 150 mm, 1.7 mm particle size, column temperature 35 ° C ~ 45 ° C; injection volume 4 ~ 10 μL; elution conditions are gradient elution: flow rate 0.2 ~ 0.5 mL / min, conditions are: 0 min, 80% ~ 90% A; 4 ~ 6 min, 75% ~ 80% A; 6 ~ 9 min, 70% ~ 75% A; 9 ~ 12 min, 65% ~ 70% A; 12 ~ 15 min, 25 ~ 35% A, 15 min ~ 17 min, 75 ~ 85% A, hold 2 ~ 4 min.

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