Detection method for quantitative urine steroid hormone based on efficient enzymolysis

Abstract

The invention relates to the technical field of analysis and detection, in particular to a detection method for quantitative urine steroid hormone based on efficient enzymolysis. The invention establishes a detection method for accurately quantifying 35 steroid hormones in urine by combining a high performance beta glucuronide enzymatic dissociation method with high performance liquid chromatography-tandem mass spectrometry. According to the method, the pretreatment time required by clinical determination of urine steroid hormones can be remarkably shortened, single-needle quantitative detection of 35 steroid hormones is realized, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a detection method for quantitative urine steroid hormones based on efficient enzymatic hydrolysis. Background Art

[0002] Steroid hormones, commonly known as steroid hormones, are a class of small molecules with a cyclopentanepolyhydrophenanthrene nucleus, synthesized and secreted by steroidogenic tissues and organs. Their molecular weight ranges from 200 to 400 Da. Based on their biological effects, they can be divided into three major categories: mineralocorticoids, glucocorticoids, and sex hormones (androgens, estrogens, and progestins). Steroid hormones play important roles in promoting biological development, maintaining normal physiological activities, and immune function. Studies have reported that a variety of diseases are associated with disorders in the synthesis and secretion of endogenous steroid hormones, such as adrenocortical carcinoma, Cushing's syndrome, and congenital adrenal hyperplasia. Therefore, timely measurement of endogenous steroid hormones is crucial for the early diagnosis and differential diagnosis of endocrine diseases.

[0003] However, although the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY bioinformatics database (https: / / www.genome.jp / pathway / map00140) provides information on at least 98 steroid hormones, and the LipidMaps database (https: / / www.lipidmaps.org / ) provides information on over 300 steroid hormones and their derivatives, the number of steroid hormones that can be accurately quantified in the laboratory is very limited. Currently, literature reports using high-performance liquid chromatography tandem mass spectrometry (LC-MS / MS) to quantitatively measure urinary steroid hormones have ranged from 1 to 26. Furthermore, steroid hormones are mostly present in the prototype form in peripheral blood. After metabolism in the liver and kidneys, they are gradually reduced and inactivated or conjugated to glucuronic acid before being excreted in the urine, which complicates their detection. Therefore, compared with the determination of plasma steroid hormones, the determination of steroid hormones in urine requires prior glucuronidase hydrolysis treatment to dissociate glucuronic acid conjugates, increase the types of prototype hormones that can be measured, and improve the detectable concentration.

[0004] Targeted multi-steroid hormone analysis based on high-performance liquid chromatography-tandem mass spectrometry presents additional challenges compared to single steroid hormone analysis. Effectively distinguishing isomers and ensuring that different hormone ion channels do not interfere with each other are the greatest challenges in LC-MS / MS method development. Therefore, utilizing LC-MS / MS to simultaneously detect a wider range of urinary steroid hormones has become a pressing technical challenge in this field. Summary of the Invention

[0005] To address the above technical difficulties, the present invention provides a method for simultaneously quantifying 35 urinary steroid hormones based on efficient enzymatic hydrolysis pretreatment, comprising: mixing a urine sample to be tested with IMCS RT enzyme and IMCS RT buffer, followed by incubation; then performing solid-phase extraction using an HLB solid-phase extraction plate to obtain an eluate; and finally detecting the eluate using high-performance liquid chromatography-tandem mass spectrometry. The conditions of the high-performance liquid chromatography-tandem mass spectrometry method include: The chromatographic column was an ACQUITY UPLC HSS C18 column with specifications of 1.8 μm and 2.1 × 150 mm. The mobile phase consisted of water and acetonitrile at a flow rate of 0.35–0.45 mL / min. Gradient elution was employed, with the sum of the volume percentages of water and acetonitrile being 100% during the gradient elution process. The gradient elution conditions were as follows: 0–1.0 min, 15% acetonitrile; 1.0–22.0 min, 15%–55% acetonitrile; 22.0–24.0 min, 55%–95% acetonitrile; 24.0–24.01 min, 95%–15% acetonitrile; and 24.01–25.0 min, 15% acetonitrile.

[0006] Preferably, the urine sample to be tested contains a hormone internal standard.

[0007] Preferably, the volume ratio of the urine sample to be tested, the IMCS RT enzyme, and the IMCS RT buffer is (5-10):1:(25-35).

[0008] Preferably, the incubation is performed at room temperature; and / or the incubation time is 0.5 h to 4 h, preferably 0.25 h to 0.5 h.

[0009] Preferably, the solid phase extraction step comprises: sequentially activating the HLB solid phase extraction plate with methanol and water, then adding the incubated mixed solution, sequentially eluting with acetonitrile water (v / v, 1:9) solution and n-hexane, and finally eluting with acetonitrile water solution (v / v, 9:1) to obtain an eluate.

[0010] Preferably, n-hexane is rinsed for 2 or more times.

[0011] Preferably, the conditions of the high performance liquid chromatography tandem mass spectrometry method further include: using a Waters Acquity I-Class high performance liquid chromatography tandem TQS triple quadrupole mass spectrometry system and adopting a multiple reaction monitoring mode to collect mass spectrometry data.

[0012] Preferably, the ion source parameters include: capillary voltage: 2~3 kV, cone voltage: 25~35 V, desolvation gas temperature: 540~560 °C, desolvation gas flow rate: 1000~1200 L / h, injection cone gas flow rate: 140~160 L / h.

[0013] Preferably, the column temperature of the chromatographic column is 20°C to 30°C.

[0014] In the present invention, the 35 urinary steroid hormones are estriol, estrone, 18-oxycortisol, progesterone, 11-deoxycortisol, estradiol, 18-hydroxycortisol, dihydrotestosterone, androstenedione, dexamethasone, 18-hydroxycortisol, 11-deoxycortisol, dehydroepiandrosterone, 17-α hydroxyprogesterone, testosterone, cortisone, cortisol, 11β-hydroxybencholanolone, tetrahydrocortisol, androsterone, 11-ketobencholanolone , 3α-hydroxy-5β-androstane-17-one, 6β-hydroxyhydrocortisone, tetrahydro-11-deoxycorticosterone, α-corticosterone tetraol, tetrahydrocortisone, iso-3α-tetrahydrocortisol, 11β-hydroxyandrostane, androst-4-ene-3,11,17-trione, 3α-hydroxy-5β-androstane-17-one, 11-ketotestosterone, β-corticosterone pentaol, 11-hydroxyprogesterone, tetrahydroaldosterone, and aldosterone.

[0015] In a specific implementation process, the urine is random urine or 24-hour urine.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This study establishes a highly efficient β-glucuronidase dissociation method combined with high-performance liquid chromatography-tandem mass spectrometry for the accurate quantification of 35 steroid hormones in urine. This method significantly shortens the pretreatment time required for clinical steroid hormone determination and enables single-injection quantitative detection of up to 35 steroid hormones, demonstrating broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the mass spectrometry response changes of some steroid hormones in a randomly selected 24-hour urine sample over the enzymatic hydrolysis time (sorted by response).

[0018] Figure 2 This is a graph showing the mass spectrometry response changes of other steroid hormones in the same randomly selected 24-hour urine sample over the enzymatic hydrolysis time (sorted by response).

[0019] Figure 3 The figure shows the separation of three isomers of tetrahydrocortisol (5β-THF), iso-3α-tetrahydrocortisol (5α-THF) and α-cortolone on the BEH C8 column; Figures A to F correspond to conditions 1 to 6, respectively.

[0020] Figure 4 Figure 1 is a graph showing the separation of two isomers, androstenone (An) and 3α-hydroxy-5β-androstane-17-one (Et), on a BEH C8 column. Figures A to F correspond to conditions 1 to 6, respectively.

[0021] Figure 5 The figure shows the separation of multiple pairs of isomers on the BEH C18 column; Figures A and B correspond to condition 1, and Figures C and D correspond to tetrahydrocortisol (5β-THF), iso-3α-tetrahydrocortisol (5α-THF) and α-corticosterone tetraol, androstenone (An) and 3α-hydroxy-5β-androstane-17-one (Et) under condition 2, respectively.

[0022] Figure 6 This is a representative peak diagram of 35 steroid hormones; among them, 1 is 6β-OHF; 2 is 18OHF; 3 is 18oxoF; 4 is E3; 5 is 18OHB; 6 is ALD; 7 is THALDO; 8 is F; 9 is E; 10 is α-cortolone; 11 is β-cortolone; 12 is 5α-THF; 13 is 11KT; 14 is 5β-THF; 15 is 5β-THE; 16 is Dexamthasone; 17 is 11KA4; 18 is 11-DF; 19 is 11β-OH-ET; 20 is 11β-OHAn; 21 is 11-oxo-Et; 22 is E2; 23 is T; 24 is 11-OHP; 25 is DOC; 26 is A4; 27 is DHEA; 28 is E1; 29 is 17OHProg; 30 is DHT; 31 is 5β-THA; 32 is Et; 33 is An; 34 is PD; and 35 is PROG. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. In the embodiments provided in this specification, those without specifying specific techniques or conditions are described in accordance with the techniques or conditions described in the literature in this area, or are carried out according to product specifications. Reagents or instruments used are not specified by manufacturer and are conventional products that can be purchased through regular channels.

[0024] Example 1 This example provides a method for determining 35 urinary steroid hormones, the steps are as follows: 1. Preparation of standard products and internal standards Thirty-five steroid hormone standards were prepared as high-concentration stock solutions in chromatography-grade methanol, based on their solubility properties. A working curve of mixed standards with appropriate concentrations was prepared using a methanol / water (1 / 1, v / v) dilution series within the physiological concentration range (Table 1 below). Similarly, a stock solution of mixed internal standards was prepared using methanol / water (1 / 1, v / v) such that after sample addition, the concentration of the internal standard working solution was as shown in Table 1 below.

[0025] Table 1 Concentration ranges of 35 steroid hormone standards and internal standards 2. Detection steps (1) Pretreatment: Add 10 μL of mixed hormone internal standard, 40 μL of randomly selected urine sample, 5 μL of IMCS RT enzyme, and 155 μL of IMCS RT buffer to a 1.5 mL EP tube. Mix gently and thoroughly, then incubate at room temperature. After incubation, add 400 μL of deionized water. After thorough vortex mixing, transfer all the contents to a 96-well HLB solid phase extraction plate (Waters, Milford, MA, USA) that has been activated (filtered with 200 μL of methanol and 200 μL of water in sequence). Discard the waste liquid under positive pressure. The HLB plate was washed once with 200 μL of acetonitrile: water (1:9, v / v) and twice with n-hexane. Finally, the analytes on the HLB plate were eluted with 40 μL of acetonitrile: water (9:1, v / v) into a 700 μL 96-well sample collection plate. 60 μL of deionized water was pre-added to the collection plate. After thorough mixing, 10 μL was injected.

[0026] (2) HPLC conditions: The chromatographic column was an ACQUITY UPLC HSS C18 column with a size of 1.8 μm and 2.1 × 150 mm. The mobile phase consisted of water and acetonitrile. A gradient elution method was used, with the sum of the volume percentages of water and acetonitrile being 100%. The gradient elution conditions were as follows: 0–1.0 min, 15% acetonitrile; 1.0–22.0 min, 15%–55% acetonitrile; 22.0–24.0 min, 55%–95% acetonitrile; 24.0–24.01 min, 95%–15% acetonitrile; 24.01–25.0 min, 15% acetonitrile. The column temperature was 25°C, and the flow rate of the mobile phase was 0.40 mL / min.

[0027] (3) Mass spectrometry conditions: A Waters Acquity I-Class high-performance liquid chromatography coupled to a TQS triple quadrupole mass spectrometer was used, and multiple reaction monitoring (MRM) mode was used for mass spectrometry data acquisition. The ion source parameters were set as follows: capillary voltage: 2.5 kV, cone voltage: 30 V, desolvation temperature: 550 °C, desolvation gas flow rate: 1100 L / h, and injection cone gas flow rate: 150 L / h.

[0028] The MRM parameters are shown in Tables 2 and 3.

[0029] Table 2 MRM parameters of 35 steroid hormones Table 3 MRM parameters of 22 steroid hormone internal standards 3. Optimization of pre-treatment conditions Follow the detection steps in step 2 and set the incubation time to 0 h, 15 min, 30 min, 1 h, 2 h, and 4 h.

[0030] The mass spectrometric response changes of steroid hormones in urine samples with enzymatic hydrolysis time are as follows: Figure 1 and Figure 2 As shown in the figure, the response concentrations of most steroid hormones, such as 11β-OHAn, are low without enzymatic hydrolysis (0 hours), gradually reaching a plateau after 0.25 hours of hydrolysis and then gradually stabilizing after 0.5 hours. Therefore, the detection method of the present invention can achieve the dissociation of all hormones from glucuronic acid within 30 minutes.

[0031] 4. Optimization of HPLC conditions (1) Chromatographic column optimization a. Follow the detection procedures in step 2, but replace the column with an ACQUITY UPLC BEH C8 1.7 μm, 2.1 × 100 mm column and use the following gradient elution conditions: 0-1.0 min, 5% B; 1.0-36.0 min, 5%-90% B; 36.0-38.0 min, 90%-90% B; 38.0-38.01 min, 90%-5% B; 38.01-39.0 min, 5% B. The flow rate was 0.3 mL / min. Use the following gradient elution conditions:

[0032] Condition 1: Phase A: 10 / 90 v / v acetonitrile / water (0.1% formic acid + 0.025 mM ammonium fluoride); Phase B: pure acetonitrile (0.1% formic acid) Condition 2: Phase A: 10 / 90 v / v methanol / water (0.1% formic acid + 0.025 mM ammonium fluoride); Phase B: pure acetonitrile (0.1% FA) Condition 3: Phase A: 10 / 90 v / v acetonitrile / water (0.1% formic acid + 0.025 mM ammonium fluoride); Phase B: pure methanol (0.1% FA) Condition 4: Phase A: water (0.1% formic acid + 0.025 mM ammonium fluoride); Phase B: 50 / 50 v / v methanol / acetonitrile (0.1% formic acid) Condition 5: Phase A: water (0.1% formic acid + 0.025 mM ammonium fluoride); Phase B: 10 / 90 v / v methanol / acetonitrile (0.1% formic acid) Condition 6: Phase A: water (0.1% formic acid + 0.025 mM ammonium fluoride); Phase B: 90 / 10 v / v methanol / acetonitrile (0.1% formic acid) The separation of three isomers of tetrahydrocortisol (5β-THF), iso-3α-tetrahydrocortisol (5α-THF) and α-cortolone on BEH C8 column is shown in Figure 2. Figure 3 As shown in Figure 2, the separation of two isomers of androstenone (An) and 3α-hydroxy-5β-androstane-17-one (Et) on the BEH C8 column is as follows: Figure 4 As shown in Figure 3, it is difficult to achieve liquid phase separation of androstenone (An) and 3α-hydroxy-5β-androstane-17-one (Et) using the BEH C8 column.

[0033] b. Follow the detection procedures in step 2 and replace the chromatographic column with a CORTECS UPLC® C18 1.6μm, 2.1 × 100mm or CORTECS UPLC® C18 1.6μm, 2.1 × 150mm. The column pressure is high and may easily exceed the maximum pressure of the system.

[0034] c. Follow the detection procedures in step 2, replacing the chromatographic column with an ACQUITY UPLC BEH C18 1.7 μm, 2.1 × 100 mm, and try different mobile phase combinations and gradients to separate the isomers tetrahydrocortisol (5β-THF), iso-3α-tetrahydrocortisol (5α-THF), and α-cortolone, as well as androsterone (An) and 3α-hydroxy-5β-androstane-17-one (Et).

[0035] Condition 1: Phase A: Water (0.1% formic acid + 0.025 mM ammonium fluoride) Phase B: 90 / 10 v / v methanol / acetonitrile (0.1% formic acid) The gradient was set as follows: 0-1.0 min, 5% B; 1.0-36.0 min, 10%-90% B; 36.0-38.0 min, 90%-90% B; 38.0-38.01 min, 90%-5% B; 38.01-39.0 min, 5% B, with a flow rate of 0.30 mL / min. The separation was poor, with peaks sticking together. Figure 5 (A and B); Condition 2: Phase A: Water (0.1% formic acid) Phase B: pure methanol The gradient was set as 0-1.0 min, 10% B; 1.0-36.0 min, 10%-90% B; 36.0-38.0 min, 90%-90% B; 38.0-38.01 min, 90%-10% B; 38.01-39.0 min, 10% B, with a flow rate of 0.30 mL / min. Although the separation was good, isomeric 3α-tetrahydrocortisol (5α-THF) was interfered with by 11-hydroxytestosterone (11OHT), as shown in Figure 2. Figure 5 (C and D); The above experimental results suggest that using a high proportion of acetonitrile as the mobile phase is more conducive to the separation of isomers. At the same time, extending the column length (100mm→150mm) and increasing the flow rate (0.3ml / min→0.4mL / min) can also help achieve more ideal chromatographic separation. Therefore, after optimizing a large number of condition combinations, it was finally found that the chromatographic detection conditions in step 2 can effectively distinguish all hormones. The representative peak diagrams of 35 steroid hormones are as follows Figure 6 shown.

[0036] Example 2 This example systematically evaluated the method established in step 2 of Example 1 with reference to the Clinical and Laboratory Standards Institute (CLSI)-C62A document, and carefully evaluated the limit of detection and the lower limit of the measurement range (limit of quantitation), linearity, precision, matrix effect, spike recovery, and carryover.

[0037] 1. The limit of detection and lower limit of the measurement interval (limit of quantification) reflect the sensitivity of the method. Based on the results of at least 10 replicates of treated low-value samples, the lowest sample concentration with a signal-to-noise ratio (S / N) > 3 was defined as the limit of detection. The lowest sample concentration with an S / N > 10 and a coefficient of variation (CV) of < 20% across replicates was defined as the limit of quantification.

[0038] 2. Linearity: As previously described, prepare a mixed hormone standard containing appropriate concentrations and serially dilute to obtain 13 different concentration points. Repeat the assay 2-4 times for each concentration point. The deviation at the limit of quantification concentration point should be within ±20%, with a coefficient of variation (CV) <20%. The deviation at the remaining concentration points should be within ±15%, with a CV <20%.

[0039] The test results are shown in Table 4.

[0040] Table 4 Verification results of detection limit, quantification limit and linear range of 35 steroid hormones It can be seen that the established detection method has high sensitivity and wide linearity, can cover the measurement range of clinical samples, and meet clinical detection needs.

[0041] 3. Precision Verification: Three concentration levels (low, medium, and high) of a pooled urine quality control were tested, with five replicates for each level, across five batches. Repeatability (%) and the coefficient of variation (CV) for intra-laboratory imprecision were calculated. A CV of <20% for the low level and <15% for the medium and high levels was considered to indicate good precision. The test results are shown in Table 5.

[0042] Table 5 Precision verification results of 35 steroid hormones The established assay exhibited good intra-assay variation of 1.86-15.10% and inter-assay variation of 4.00-19.75%. Notably, the method's precision was evaluated on discrete days, with the mass spectrometer performing other measurements simultaneously. The results still met the performance validation requirements, demonstrating the robustness of the method and its suitability for routine clinical testing.

[0043] 4. Matrix Effects: After processing at least five sample matrices and four blank matrices in parallel, spike the parallel samples with mixed standards at concentrations of none, low (Level 1), medium (Level 2), and high (Level 3) to evaluate the differences in hormone response to different concentrations in the different matrices. If there is a significant difference in the response of the same hormone standard in the blank matrix and sample matrix, it indicates significant matrix suppression / enhancement. Correction for this hormone using an isotopic internal standard is recommended. A matrix effect of 85%-115% is considered acceptable. The test results are shown in Table 6.

[0044] Table 6 Matrix effect results of 35 steroid hormones As can be seen, the responses of most hormones were significantly suppressed in the urine matrix, however, this could be corrected by the internal standard. The matrix effect of the established assay met the detection requirements when properly corrected by the internal standard.

[0045] 5. Spike Recovery: Three standards of known concentrations (low, medium, and high) were spiked into a blank matrix. At least five replicates were tested for each concentration. Accuracy was evaluated by the ratio of the measured concentration to the theoretical concentration. The difference between the theoretical values ​​for the medium and high values ​​should not exceed 15%. The difference between the measured value and the low theoretical concentration should be within 20%. The test results are shown in Table 7.

[0046] Table 7 Recovery results of 35 steroid hormones spiked It can be seen that the spike recovery of the established detection method meets the performance verification requirements.

[0047] 6. Carryover contamination: The hormone residues in the blank sample after each high-concentration sample injection reflect the residual risk in the system. The results showed that the residual peak area was lower than 25% of the peak area of ​​the lower limit of the measurement reference range.

[0048] In summary, the present invention utilizes efficient β-glucuronidase dissociation pretreatment in conjunction with LC-MS / MS technology to achieve the simultaneous determination of 35 steroid hormones in urine samples, enabling the combined clinical application of multiple hormone indicators. The detection method of the present invention achieves enzymatic digestion of samples within 30 minutes, overcoming the limitation of urine samples requiring several hours of enzymatic digestion reported in previous literature and shortening the total analysis time. Furthermore, this method can achieve the effective separation of multiple pairs of isomers, minimizing the impact of potential interference on detection.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for detecting urinary steroid hormones based on efficient enzymatic hydrolysis, characterized in that: include: The urine sample to be tested is mixed with IMCS RT enzyme and IMCS RT buffer and incubated, and then solid phase extraction is performed using an HLB solid phase extraction plate to obtain an eluate, and finally the eluate is detected by high performance liquid chromatography tandem mass spectrometry; the conditions of the high performance liquid chromatography tandem mass spectrometry method include: The chromatographic column was an ACQUITY UPLC HSS C18 column with specifications of 1.8 μm and 2.1 × 150 mm. The mobile phase consisted of water and acetonitrile at a flow rate of 0.35–0.45 mL / min. Gradient elution was employed, with the sum of the volume percentages of water and acetonitrile being 100% during the gradient elution process. The gradient elution conditions were as follows: 0–1.0 min, 15% acetonitrile; 1.0–22.0 min, 15%–55% acetonitrile; 22.0–24.0 min, 55%–95% acetonitrile; 24.0–24.01 min, 95%–15% acetonitrile; and 24.01–25.0 min, 15% acetonitrile.

2. The detection method according to claim 1, wherein The urine sample to be tested contains a hormone internal standard.

3. The detection method according to claim 1, wherein The volume ratio of the urine sample to be tested, IMCS RT enzyme and IMCS RT buffer is (5-10):1:(25-35).

4. The detection method according to claim 1, wherein The incubation is performed at room temperature; and / or the incubation time is 0.5 h to 4 h, preferably 0.25 h to 0.5 h.

5. The detection method according to claim 1, wherein The solid phase extraction step includes: activating the HLB solid phase extraction plate with methanol and water in sequence, then adding the incubated mixed solution, eluting with acetonitrile aqueous solution and n-hexane in sequence, and finally eluting with acetonitrile aqueous solution to obtain an eluate.

6. The detection method according to claim 5, characterized in that Rinse with n-hexane 2 or more times.

7. The detection method according to claim 1, characterized in that The conditions of the high performance liquid chromatography tandem mass spectrometry method also include: using a Waters Acquity I-Class high performance liquid chromatography tandem TQS triple quadrupole mass spectrometry system and adopting a multiple reaction monitoring mode to collect mass spectrometry data.

8. The detection method according to claim 7, characterized in that The ion source parameters included: capillary voltage: 2–3 kV, cone voltage: 25–35 V, desolvation temperature: 540–560 °C, desolvation gas flow rate: 1000–1200 L / h, and injection cone gas flow rate: 140–160 L / h.

9. The detection method according to claim 1, wherein The column temperature of the chromatographic column is 20℃~30℃.

10. The detection method according to claim 1, characterized in that The 35 urinary steroid hormones are estriol, estrone, 18-hydroxycortisol, progesterone, 11-deoxycortisol, estradiol, 18-hydroxycortisol, dihydrotestosterone, androstenedione, dexamethasone, 18-hydroxycortisol, 11-deoxycortisol, dehydroepiandrosterone, 17-α-hydroxyprogesterone, testosterone, cortisone, cortisol, 11β-hydroxybencholanolone, tetrahydrocortisol, androsterone, 11-ketobencholanolone, 3α -hydroxy-5β-androstane-17-one, 6β-hydroxyhydrocortisone, tetrahydro-11-deoxycorticosterone, α-corticosterone tetraol, tetrahydrocortisone, iso-3α-tetrahydrocortisol, 11β-hydroxyandrostane, androst-4-ene-3,11,17-trione, 3α-hydroxy-5β-androstane-17-one, 11-ketotestosterone, β-corticosterone pentaol, 11-hydroxyprogesterone, tetrahydroaldosterone, and aldosterone.

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