Method for simultaneously detecting multiple steroid hormones in serum

Abstract

The invention relates to a method for simultaneously detecting various steroid hormones in serum, which comprises the following steps: extracting hormones in serum by a liquid-liquid extraction method; a derivatization reagent is added into the extracted hormone, an oximation reaction is carried out, and the derivatization reagent comprises quaternary ammonium oxygen amine; the hormone after oximation reaction is injected into a two-dimensional liquid chromatography-tandem mass spectrometry system for detection, the hormone is enriched on a one-dimensional chromatographic column, and then the enriched hormone is subjected to back flushing and transferred to a two-dimensional chromatographic column for analysis. According to the method, the backwashing transfer two-dimensional liquid chromatography-tandem mass spectrometry and the derivatization reaction are combined, various steroid hormones including progestational hormone and androgen can be detected at the same time, and the method has the advantage of being high in detection sensitivity.

Description

Technical Field

[0001] This application relates to a detection method using liquid chromatography coupled with tandem mass spectrometry, and more particularly to a method for simultaneously detecting multiple steroid hormones in serum. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disorder affecting women of reproductive age, impacting approximately 8%-13% of women worldwide and becoming a leading cause of female infertility. Hyperandrogenemia is a hallmark feature of PCOS, with elevated androgen levels often accompanied by insulin resistance, ovulation disorders, and other endocrine and metabolic disturbances. Currently, the sources and mechanisms of excessive androgen secretion in PCOS patients are not fully understood, and significant individual differences in sex hormone profiles exist among patients, leading to varying clinical treatment plans. Therefore, accurate quantitative detection of sex hormones in PCOS patients is of significant clinical importance.

[0003] Sex hormones are an important class of steroid hormones in the human body, mainly including progesterone, androgens, and estrogens. During the fetal period and before puberty, the adrenal glands play a crucial role in sex hormone synthesis; after puberty, the main site of sex hormone synthesis shifts to the gonads. In clinical practice, the main sex hormones tested for patients with polycystic ovary syndrome (PCOS) include testosterone (T), androstenedione (A4), progesterone (P), and estradiol (E2), with testosterone and androstenedione levels often showing significant elevations. However, the human body contains a wide variety of sex hormones, and the interconversion relationships between these hormones are extremely complex. Relying solely on testing these few hormones is insufficient to accurately and comprehensively reflect the sex hormone profile of PCOS patients. In the past decade, increasing research has revealed that 11-oxidized androgens play a crucial role in PCOS and other diseases. In the human body, both 11-oxidized androgens and classical androgens use cholesterol and progesterone as upstream precursors for synthesis. Progesterone is first converted to classical androgens, which are then further converted to 11-oxidized androgens under the action of the CYP11B enzyme. These 11-oxidized androgens are expected to become important biomarkers for the diagnosis of polycystic ovary syndrome (PCOS). However, research on the detection of 11-oxidized androgens in PCOS patients is still relatively scarce.

[0004] In clinical practice, immunoassay remains the standard method for sex hormone detection. While this method offers advantages such as high automation and low cost, its inherent limitations affect the accurate quantification of various hormones. First, structurally similar steroid hormones may cross-react with detection antibodies, leading to positive biases in the results and decreased analytical precision. Second, the levels of many circulating sex hormones in the human body (such as dihydrotestosterone and 11-hydroxytestosterone) are extremely low, often below the reliable quantitative sensitivity limit of immunoassay.

[0005] To overcome the limitations of immunoassays in terms of specificity, sensitivity, and simultaneous detection of multiple indicators, the *Journal of Clinical Endocrinology and Metabolism* explicitly proposed in 2013 that studies with sex hormone detection as the primary endpoint should employ mass spectrometry-based detection methods. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) quantifies compounds based on molecular mass and characteristic fragment ions, offering advantages such as high specificity, high sensitivity, simultaneous analysis of multiple components, and no cross-reaction interference. To further improve separation efficiency and detection sensitivity, two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS / MS) has emerged. Its basic principle involves connecting two chromatographic columns via a switching valve. The fraction eluted from the one-dimensional column is collected after multiple cycles and injected into the two-dimensional column for further separation. This technique exhibits excellent performance in terms of separation capability and information acquisition. Currently, two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS / MS) has been widely used in the analysis of natural medicines, proteomics, and separation of chiral compounds. However, its application in the analysis of steroid hormones remains limited and has significant limitations. For example, Adriaansen BPH et al. used 2D-LC-MS / MS to determine 11-oxygenated androgens in children's serum, but their sample pretreatment method used solid-phase extraction, which was cumbersome and expensive. Furthermore, the chromatographic conditions used in this method, such as setting the column temperature beyond the conventional range, could significantly damage the column. Additionally, the multiple variable-speed rinsing of the mobile phase during the analysis of a single sample could easily cause the retention time of the analyte to drift. These chromatographic conditions, being too extreme, make it difficult to guarantee the stability of long-term detection. (Adriaansen BPH, Oude Alink SE, Swinkels DW, et al. Reference intervals for serum 11-oxygenated androgens in children. European Journal of Endocrinology, 2024, 190(1):96-103). Summary of the Invention

[0006] To address the problems existing in the prior art, this application provides a method for simultaneously detecting multiple steroid hormones in serum. The method of this application combines reverse washing transfer two-dimensional liquid chromatography-tandem mass spectrometry with derivatization reaction, which can simultaneously detect multiple steroid hormones, including progesterone and androgens, and has the advantage of high detection sensitivity.

[0007] Item 1. A method for simultaneously detecting multiple steroid hormones in serum, wherein the method comprises: Hormones were extracted from serum using liquid-liquid extraction. A derivatization reagent is added to the extracted hormone to carry out an oxime reaction, wherein the derivatization reagent contains quaternary ammonium oxyamine; The hormone after oxime reaction is injected into a two-dimensional liquid chromatography-tandem mass spectrometry system for detection. The hormone is enriched on a one-dimensional chromatographic column, and then the enriched hormone is backwashed and transferred to a two-dimensional chromatographic column for analysis.

[0008] Item 2, the method according to Item 1, wherein the packing material of the two-dimensional chromatographic column comprises adamantyl and / or phenylhexyl.

[0009] Item 3. The method according to Item 2, wherein the volume ratio of adamantyl and phenylhexyl in the two-dimensional chromatographic column is 4:1.

[0010] Item 4. According to the method of Item 1, wherein during detection, a mixed mobile phase consisting of mobile phase A and mobile phase B is used for gradient elution, wherein mobile phase A is an aqueous solution of ammonium formate with a concentration of 10 mM containing 0.1% (v / v) formic acid, and mobile phase B is acetonitrile containing 0.1% (v / v) formic acid; During gradient elution, the volume percentage of mobile phase B in the mixed mobile phase gradually increases from the initial percentage and then returns to the initial percentage. During gradient elution, the elution temperature was 45℃, the flow rate was 0.6 mL / min, and the elution time was 21 min.

[0011] Item 5. The method according to Item 4, wherein, when the hormone is enriched on the one-dimensional chromatographic column, the mobile phase A is also delivered to the one-dimensional chromatographic column via an auxiliary pump.

[0012] Item 6. The method according to Item 1, wherein the total time for the backwash transfer is 0.1-5 min.

[0013] Item 7. The method according to Item 1, wherein the derivatizing reagent further comprises methanol, water and formic acid, wherein the concentration of formic acid is 2-15% (V / V).

[0014] Item 8. According to the method described in Item 1, the reaction temperature of the oxime reaction is 30-50℃ and the reaction time is 60-90min.

[0015] Item 9. The method according to Item 3, wherein the one-dimensional chromatographic column is a C18 chromatographic column with dimensions of 100×4.6mm and 5μm; and the two-dimensional chromatographic column has dimensions of 100×3mm and 2.6μm.

[0016] Item 10. The method according to Item 1, wherein the steroid hormone is selected from testosterone, dehydroepiandrosterone, dihydrotestosterone, androstenedione, 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketotestosterone, 11-ketoandrostenedione, progesterone, 17-hydroxyprogesterone, pregnenolone, and 17-hydroxypregnenolone.

[0017] Beneficial effects 1. The detection method of this application increases the injection volume from 5 μL to 40 μL. Without producing obvious solvent effects, deteriorating peak shape, or significantly increasing matrix interference, it significantly increases the absolute amount of analyte injected and the detection sensitivity, reduces the need for sample pretreatment concentration, broadens the scope of application of the method, and improves the accuracy and stability of trace component quantification.

[0018] 2. The detection method of this application does not require complex protein removal, impurity purification, and sample pre-concentration. Simple pretreatment alone can significantly reduce matrix and protein interference, greatly improving detection sensitivity and throughput. The pretreatment step of this application uses liquid-liquid extraction, which is simple to operate and can significantly save serum sample volume, reduce sample loss, and improve sample utilization. Even in the presence of impurities and potential protein interference in the sample, the detection method described in this application can still significantly improve the sensitivity of hormone detection.

[0019] 3. The detection method of this application can simultaneously detect total androgens and progesters in serum with high sensitivity, and can simultaneously detect multiple hormones, which can reduce the number of sample pretreatments, reduce sample loss, and improve detection throughput and data reliability.

[0020] 4. The detection method of this application combines chemical derivatization with reverse-flushing transfer two-dimensional liquid chromatography-tandem mass spectrometry, which significantly improves the sensitivity of mass spectrometry detection. The established method has a quantitation limit of picograms per milliliter for a variety of sex hormones, and is suitable for accurate quantitative analysis of trace sex hormones in healthy people, diseased people and special populations.

[0021] 5. Compared with the prior studies of our research team (CN202211366715X and CN2025101044132), the detection method of this application can detect a wider variety of sex hormones. More importantly, the detection method of this application greatly reduces the lower limit of quantification of sex hormones in serum. Compared with the prior studies, the lower limit of quantification is reduced by 5 to 10 times. This means that the method has a wider applicability and can meet the detection needs of certain specific populations (such as postmenopausal women, children, etc.). Attached Figure Description

[0022] Figure 1 It refers to the synthesis and metabolism pathways of progesterone and androgens in the human body.

[0023] Figure 2In the diagram, A is a schematic diagram of the basic principle of reverse flushing transfer two-dimensional liquid chromatography-tandem mass spectrometry, and B is the chemical reaction equation of quaternary ammonium oxyamine-derived steroid hormones.

[0024] Figure 3 These are the mass spectrometry conditions for 12 sex hormones and 8 isotope internal standards.

[0025] Figure 4 This shows the separation effect of different two-dimensional chromatographic columns on specific isomers. A: Agilent Poroshell EC-C18 column (100×3.0 mm, 2.7 μm); B: Demeter Adamantyl column (100×3.0 mm, 3.0 μm); C: KF5-W column (100×3.0 mm, 2.6 μm).

[0026] Figure 5 The peak shapes of 11OHA4-QAO and 11OHT-QAO are shown using different one-dimensional chromatographic columns with an injection volume of 40 μL. A: Demeter C18 column (50 × 3.0 mm, 3.5 μm); B: Waters C18 column (100 × 4.6 mm, 5 μm). It can be observed that when using a shorter column, the peak shape is poor and bifurcation is more likely to occur.

[0027] Figure 6 It is the backflushing transfer time window of the analyte on a one-dimensional chromatographic column: starting from 1.5 min and ending at 4.75 min. Figure 7 It is a chromatogram of one-dimensional and two-dimensional chromatographic columns connected in series, with an injection volume of 40 μL.

[0028] Figure 8 This involves optimizing the derivatization conditions of quaternary ammonium oxide. A: Optimization of quaternary ammonium oxide dosage; B: Optimization of formic acid dosage; C: Optimization of derivatization temperature; D: Optimization of derivatization time.

[0029] Figure 9 The calibration range, linearity, inter-batch and intra-batch accuracy and precision of 12 hormone substances were determined using the detection method of this application.

[0030] Figure 10 The matrix effect and extraction recovery rate of the detection method in this application are shown.

[0031] Figure 11-14 It is the result of the matrix effect of different substances.

[0032] Figure 15 This is to assess the stability and dilution reliability of the detection method in this application.

[0033] Figure 16The baseline clinical characteristics of individuals with and without polycystic ovary syndrome (PCOS) and those with PCOS.

[0034] Figure 17 The results of serum steroid hormone tests are for individuals with and without polycystic ovary syndrome (PCOS) and individuals with PCOS.

[0035] Figure 18 This study compared the differences in serum levels of various steroid hormones between individuals with and without polycystic ovary syndrome (PCOS). ns: No significant difference; **: p ≤0.01; ***: p ≤0.001.

[0036] Figure 19 The chromatograms are of low concentrations of various analytes in actual samples detected using the detection method of this application; 11KT (0.203 ng / mL); 11OHA4 (0.482 ng / mL); 11OHT (0.088 ng / mL); 17OHP (0.299 ng / mL); 17OHPreg (1.273 ng / mL); A4 (0.379 ng / mL); DHEA (0.644 ng / mL); DHT (0.029 ng / mL); P (0.127 ng / mL); Preg (0.407 ng / mL); T (0.071 ng / mL); 11KA4 (0.571 ng / mL).

[0037] Figure 20 This study compares the quantitative steroid hormone studies based on LC-MS / MS over the past five years with the detection method described in this application.

[0038] Figure 21 This is a schematic diagram of the detection method steps of this application. First, serum samples from individuals with polycystic ovary syndrome are collected. After appropriate pretreatment, a derivatization reaction is performed, followed by separation and detection by two-dimensional liquid chromatography-tandem mass spectrometry after reverse washing and transfer.

[0039] Figure 22 This is the setup used by Adriaansen et al. to determine classic androgens. Mobile phase A: water + 0.1 mM ammonium fluoride + 0.1% formic acid; Mobile phase B: 100% methanol. RT: retention time; MRM: multiple reaction monitoring; CE: collision energy.

[0040] Figure 23 This is the setup used by Adriaansen et al. to determine 11-oxidized androgens. Mobile phase A: water + 0.1 mM ammonium fluoride + 0.1% formic acid; Mobile phase B: 100% methanol. RT: retention time; MRM: multiple reaction monitoring; CE: collision energy.

[0041] Figure 24 Adriaansen et al. measured the median androgen concentration in children of different ages.

[0042] Figure 25 This is a representative chromatogram of A: classical androgens and B: 11-oxidized androgens in human serum samples determined by Adriaansen et al. Detailed Implementation

[0043] Specific embodiments of this application will now be described in more detail. While the following description illustrates specific embodiments of this application, it should be understood that this application can be implemented in various forms and should not be limited to the specific embodiments or implementations set forth herein. Rather, these specific embodiments or implementations are provided to enable a more thorough understanding of this application and to fully convey the scope of this application to those skilled in the art.

[0044] In this application, the term "steroid hormone" refers to a class of steroid hormones with a cyclopentane-polyhydrophenanthrene core structure. In this application, "steroid hormone" and "steroid hormone" are used interchangeably.

[0045] In this application, the term "liquid-liquid extraction" refers to a sample pretreatment technique in which an organic solvent immiscible with water is added to a biological sample as an extraction solvent, and after vortex mixing and centrifugation to separate the sample layers, the target hormone is transferred from the aqueous sample matrix to the organic phase extraction liquid, thereby removing matrix interference such as proteins and salts, and achieving the extraction, purification and enrichment of the target substance.

[0046] In this application, the quaternary ammonium oxyamine derivatizing agent refers to an aminooxy group that can react with the carbonyl group of steroids. In this application, "quaternary ammonium oxyamine" and "QAO" can be used interchangeably.

[0047] In this application, the terms "derivative reaction" and "oxime reaction" are used interchangeably.

[0048] In this application, the term "two-dimensional liquid chromatography" refers to the online series connection of a first chromatographic column and a second chromatographic column through an interface such as a switching valve, where the sample to be tested is first preliminarily separated, enriched or purified on the first chromatographic column, and then the target components are transferred online to the second chromatographic column for further fine separation.

[0049] In this application, "%(v / v)" represents the volume percentage of the substance. For example, "the concentration of the methanol solution is 80%(v / v)" means that the volume percentage of methanol in the solution is 80%, that is, methanol accounts for 80 volume units in every 100 volume units of the solution.

[0050] This application provides a method for simultaneously detecting multiple steroid hormones in serum. In some embodiments, the steroid hormones are selected from testosterone, dehydroepiandrosterone, dihydrotestosterone, androstenedione, 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketotestosterone, 11-ketoandrostenedione, progesterone, 17-hydroxyprogesterone, pregnenolone, and 17-hydroxypregnenolone. The detection method of this application includes the following steps.

[0051] Step 1: Extract hormones from serum. Specifically, extract hormones from serum using liquid-liquid extraction.

[0052] In some implementations, the liquid-liquid extraction method uses a mixed solution of tert-butyl methyl ether and ethyl acetate as the extraction solvent.

[0053] In some embodiments, the volume ratio of tert-butyl methyl ether to ethyl acetate is (3-7):(7-3). Specifically, the volume ratio of tert-butyl methyl ether to ethyl acetate can be 3:7, 4:6, 5:5, 6:4, or 7:3.

[0054] In some embodiments, the volume ratio of tert-butyl methyl ether to ethyl acetate is 5:5.

[0055] In some embodiments, during the extraction process, the volume ratio of serum to the extraction solvent is 1:(2-8). Specifically, the volume ratio of serum to the extraction solvent can be 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8.

[0056] In some embodiments, the volume ratio of serum to extraction solvent is 1:5 during the extraction process.

[0057] Step two, a derivatization reagent is added to the extracted hormone to carry out an oxime reaction, wherein the derivatization reagent contains quaternary ammonium oxide.

[0058] The derivatization reagent containing quaternary ammonium oxyamine (QAO) used in this application has a permanently charged mass spectrometry tag and an amino group that can react with steroid carbonyl groups, thereby greatly improving ionization efficiency (complete ionization). This allows the method described in this application to detect low-abundance androgens, including those that are undetectable by electrochemiluminescence immunoassay (ECLIA). In other words, the QAO-containing derivatization reagent used in this application significantly improves the sensitivity of mass spectrometry. This method enables the detection of multiple androgens even in laboratories equipped with lower-end mass spectrometry systems, providing significant assistance for disease diagnosis and management.

[0059] This application uses a derivatizing reagent containing quaternary ammonium oxychloride for in-situ derivatization, essentially utilizing an oxime reaction to derivatize androgens to obtain oxime-formed androgen products. Those skilled in the art should understand that any quaternary ammonium oxychloride derivatizing reagent containing an amino group can achieve this application, regardless of the anion bonded to the amino group, such as QAO ⇌ Br or Cl in QAO halides, where the Br or Cl ions do not affect the oxime-formation of androgens.

[0060] In some embodiments of this application, the derivatizing agent includes QAO halides.

[0061] In some embodiments, the concentration of quaternary ammonium oxide in the derivatizing reagent is 0.01-0.5 mol / L, specifically, it can be 0.01 mol / L, 0.05 mol / L, 0.10 mol / L, 0.15 mol / L, 0.20 mol / L, 0.25 mol / L, 0.30 mol / L, 0.35 mol / L, 0.40 mol / L, 0.45 mol / L, or 0.50 mol / L.

[0062] In some embodiments, the concentration of quaternary ammonium oxychloride in the derivatizing reagent is 0.1-0.2 mol / L.

[0063] In some embodiments, the concentration of quaternary ammonium oxide in the derivatizing reagent is 0.1 mol / L.

[0064] In some embodiments, an oxime reaction is carried out by adding a derivatizing reagent containing 1-20 μmol of quaternary ammonium oxychloride to each 100 μL of hormone extracted from serum. Specifically, the derivatizing reagent may contain 1 μmol, 2 μmol, 3 μmol, 4 μmol, 5 μmol, 6 μmol, 7 μmol, 8 μmol, 9 μmol, 10 μmol, 11 μmol, 12 μmol, 13 μmol, 14 μmol, 15 μmol, 16 μmol, 17 μmol, 18 μmol, 19 μmol, or 20 μmol of quaternary ammonium oxychloride.

[0065] In some embodiments, an oxime reaction is carried out by adding a derivatizing reagent containing 5-15 μmol of quaternary ammonium oxyamine to each 100 μL of hormone extracted from serum.

[0066] In some embodiments, an oxime reaction is carried out by adding a derivatization reagent containing 10 μmol of quaternary ammonium oxyamine to each 100 μL of hormone extracted from serum.

[0067] In some implementations, the derivatizing agent further comprises methanol and water.

[0068] In some embodiments, the derivatizing agent further comprises formic acid, wherein the concentration of formic acid is 2-15% (V / V), specifically, it can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%.

[0069] In some embodiments, the concentration of formic acid in the derivatizing agent is 8% (V / V).

[0070] In some embodiments, the reaction temperature of the oxime reaction is 20-60°C, specifically, it can be 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C.

[0071] In some embodiments, the oxime reaction is carried out at a temperature of 40-50°C.

[0072] In some embodiments, the oxime reaction is carried out at a temperature of 40°C.

[0073] In some embodiments, the reaction time of the oxime reaction is 30-120 min, specifically, it can be 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, 95 min, 100 min, 105 min, 110 min, 115 min, or 120 min.

[0074] In some implementations, the oxime reaction takes 60-90 minutes.

[0075] In some embodiments, the oxime reaction takes 75 minutes.

[0076] Step 3: The hormone after oxime reaction is injected into a two-dimensional liquid chromatography-tandem mass spectrometry system for detection. The hormone is enriched on a one-dimensional chromatographic column, and then the enriched hormone is backwashed and transferred to a two-dimensional chromatographic column for analysis.

[0077] This application employs two-dimensional liquid chromatography for the enrichment and separation of hormones, and its separation capability, selectivity, sensitivity, or resistance to matrix interference are significantly superior to the chromatographic separation techniques of single-dimensional liquid chromatography.

[0078] In some implementations, the one-dimensional chromatographic column is an octadecylsilane-bonded porous silica column.

[0079] Those skilled in the art will understand that any brand of octadecylsilane-bonded porous silica column can be used in the scheme of this application. The only difference lies in the elution process during high-performance liquid chromatography (HPLC) separation due to variations between different brands of columns. The columns include, but are not limited to, commercially available core-shell octadecylsilane-bonded columns from manufacturers such as Agilent, Waters, and Thermo. Specifically, the columns can be ACQUITY UPLC BEH C18, Poroshell EC-C18, or Poroshell EC-C18.

[0080] In some embodiments, the one-dimensional chromatographic column is preferably a Waters C18 column with dimensions of 100×4.6mm and 5μm.

[0081] In some embodiments, the two-dimensional chromatographic column is an adamantyl alkyl chromatographic column.

[0082] In some embodiments, the packing material in the two-dimensional chromatographic column further includes phenylhexyl groups.

[0083] In some embodiments, the packing material in the two-dimensional chromatographic column is adamantyl and phenylhexyl, wherein the volume ratio of adamantyl to phenylhexyl is 4:1.

[0084] In some embodiments, the two-dimensional chromatographic column has dimensions of 100 × 3 mm and a diameter of 2.6 μm. In some embodiments, during chromatographic separation, the mixed mobile phase for gradient elution consists of mobile phase A and mobile phase B. The gradient elution process using the mixed mobile phase involves gradually increasing the volume percentage of mobile phase B in the mixed mobile phase from an initial percentage, and then restoring it to the initial percentage.

[0085] Those skilled in the art will know that any mobile phase that enables the test sample to achieve sufficient separation and good peak shape can be used in the scheme of this application.

[0086] In some embodiments, gradient elution is performed in both one-dimensional and two-dimensional chromatographic columns using a mixed mobile phase consisting of mobile phase A and mobile phase B, wherein mobile phase A is a 10 mM aqueous solution of ammonium formate containing 0.1% (v / v) formic acid, and mobile phase B is acetonitrile containing 0.1% (v / v) formic acid. During gradient elution, the volume percentage of mobile phase B in the mixed mobile phase gradually increases from the initial percentage and then returns to the initial percentage.

[0087] In gradient elution, the volume percentage of mobile phase B in the mixed mobile phase can be gradually increased initially for analyte elution, then increased to a high percentage for column cleaning, and finally returned to initial equilibrium. This process can be repeated during analysis. Those skilled in the art should understand that the change in the ratio of A to B in the mobile phase during gradient elution is a continuous process, and depending on the specific implementation of the gradient elution, those skilled in the art can allow the phase to remain at any ratio for a certain period of time, or adjust the rate of change between any two ratios, based on the operating conditions at the time.

[0088] In some implementations, the gradient elution process is as follows (all proportions mentioned in the elution process are volume proportions).

[0089] Stage 1: The initial proportion of mobile phase B is 1-20%, specifically, the proportion can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%.

[0090] Phase 2: Based on the properties of the analytes in the sample, the proportion of mobile phase B is gradually increased in multiple stages over a certain period of time. For example, within 10-40 minutes, the proportion of mobile phase B is increased to 20-100% in 3-10 stages, allowing analytes of different polarities in the sample to elute sequentially, achieving better separation and enabling accurate quantification. Specifically, the proportion of mobile phase B can be increased to 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% in stages 3, 4, 5, 6, 7, 8, 9, and 10, respectively.

[0091] Stage 3: After the analytes have been mostly eluted, mobile phase B is maintained at 85-100% for a certain period of time to thoroughly wash away impurities in the column, ensuring that even weakly polar substances in the sample are completely eluted, thus not affecting the next injection. For example, mobile phase B can be maintained at 85%, 90%, 95%, or 100% for 1-10 minutes. Specifically, it can be maintained for 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, 6.5 minutes, 7 minutes, 7.5 minutes, 8 minutes, 8.5 minutes, 9 minutes, 9.5 minutes, or 10 minutes.

[0092] Phase 4: The proportion of mobile phase B is restored to its initial proportion, i.e., the proportion of mobile phase B in Phase 1, and maintained for a certain period of time to achieve equilibration, in order to ensure the reproducibility of each injection. For example, mobile phase B is maintained at the proportion of Phase 1 for 1-10 minutes, specifically, for 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min, 6 min, 6.5 min, 7 min, 7.5 min, 8 min, 8.5 min, 9 min, 9.5 min, and 10 minutes.

[0093] In some implementations, the gradient elution process is as follows (all proportions mentioned in the elution process are volume proportions).

[0094] Stage 1: The initial volume percentage of mobile phase B is 15%; Phase 2: Over 16.1 minutes, the volume percentage of mobile phase B is gradually increased to 100% in 5 stages. Stage 3: The volume of mobile phase B is 100%, and it is maintained for 2.9 min; Phase 4: Within 0.1 min, reduce the volume percentage of mobile phase B to 15% and maintain this for 2.9 min.

[0095] In some implementation schemes, the time and volume ratio of mobile phase B in each stage of the above implementation scheme can be adjusted according to the actual situation.

[0096] In some implementations, the gradient elution process is as follows (all proportions mentioned in the elution process are volume proportions).

[0097] The initial proportion of mobile phase B is 15%. Within 0-10 minutes, the proportion of mobile phase B increased to 28%; After 10-10.5 minutes, the proportion of mobile phase B increases to 30%. 10.5-11.5 min, maintain the proportion of mobile phase B at 30%; 11.5-16 min, the proportion of mobile phase B increases to 40%; 16-16.1 min, the proportion of mobile phase B increases to 100%; 16.1–19 min, the proportion of mobile phase B is maintained at 100%; At 19.1 min, the proportion of mobile phase B was reduced to 15% and maintained for 21 min to equilibrate the column.

[0098] In some implementations, when the hormone is enriched on a one-dimensional chromatographic column, the mobile phase A is also delivered to the one-dimensional chromatographic column.

[0099] In some implementations, the mobile phase A is also delivered to the one-dimensional chromatographic column via an auxiliary pump for 0.1-0.7 min.

[0100] In some embodiments, during the elution process, the one-dimensional column is backwashed to transfer the analyte enriched in the one-dimensional column to the two-dimensional column. Backwashing refers to the operation of flowing the mobile phase through the column in the opposite direction to that in conventional analysis. In this application, during the backwashing transfer process, only the flow direction of the mobile phase is changed, without altering the proportion of the mobile phase.

[0101] In some implementations, the total backwash transfer time is 0.1-5 min. Specifically, the total backwash transfer time can be 0.1 min, 0.5 min, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, or 5 min.

[0102] In some implementations, during the elution process, the one-dimensional column is backwashed and transferred at 1.5–4.75 min to elute the analytes enriched in the one-dimensional column to the two-dimensional column, where the analytes are detected and analyzed.

[0103] In some implementations, during gradient elution, the elution temperature is 45°C and the flow rate of the mobile phase is 0.6 mL / min.

[0104] In some implementations, the injection volume is 40 μL.

[0105] Example Material sources used in the embodiments Progesterone (P), Pregnenolone (Preg), 17-hydroxyprogesterone (17OHP), 17-hydroxypregnenolone (17OHPreg), deuterated progesterone-d9 (P-d9), deuterated pregnenolone-d4 (Preg-d4), carbon 13 labeled 17-hydroxyprogesterone- 13 C3(17OHP- 13C3 and deuterated 17-hydroxypregnenolone-d3 (17OHPreg-d3) were purchased from Alta Scientific Co., Ltd (Tianjin, China); testosterone was purchased from European Pharmacopoeia; androstenedione was purchased from Macklin Biochemical Co., Ltd (Shanghai, China); 11-ketotestosterone and dihydrotestosterone were purchased from CFW Laboratories, USA; 11-hydroxytestosterone, 11-hydroxyandrostenedione (11OHA4), and 11-ketoandrostenedione (11KA4) were all purchased from Steraloids Inc. (Newport, RI, USA); deuterated 11-hydroxyandrostenedione-d4 and deuterated testosterone-d3 were purchased from Cambridge Isotope Laboratories Inc. (Andover, MA, USA). All standards and internal standards had a purity of not less than 96%. Quaternary ammonium oxide was synthesized by Kanglong Chemical Co., Ltd., with a purity of 97.5%. Mass spectrometry grade acetonitrile, methanol, ethyl acetate, tert-butyl methyl ether, and formic acid were purchased from Fisher Scientific (Fair Lawn, NJ, USA); ammonium formate was purchased from Sigma-Aldrich Co. (St Louis, MO, USA); and ultrapure water was prepared in the laboratory.

[0106] Blank substrate All target hormones detected in this study were endogenous substances; therefore, standard serum and quality control samples could not be prepared using ordinary serum. To assess matrix effects and method specificity, method validation required serum from six different individuals. This study used activated carbon adsorption to prepare blank serum: human serum and activated carbon were mixed at a ratio of 20:1 (volume / mass), vortexed for 5 minutes, incubated at 4°C for 48 hours, then centrifuged at 15,000 rpm for 10 minutes, and filtered through a 0.2-micron filter to obtain steroid hormone-free blank serum. This method is a well-validated and classic approach in steroid hormone analysis, minimizing interference with the detection results. In liquid-liquid extraction condition optimization and other method validation experiments, commercially available hormone-free human serum purchased from Shanghai Pufen Biotechnology Co., Ltd. was used.

[0107] Preparation of standards, calibrators and quality control sample solutions Dissolve each standard separately in methanol to prepare a primary stock solution with a concentration of 1 mg / mL. Take an appropriate amount of the primary stock solution and dilute it with methanol to prepare a secondary stock solution with a concentration of 10 μg / mL. Mix the corresponding volumes of the secondary stock solutions of each analyte, and then dilute with methanol to prepare a mixed standard stock solution, wherein the concentration of each analyte is 10 times that of its upper limit of quantitation working solution. Perform serial dilutions of the mixed standard stock solution to prepare calibration working solutions with 8 concentrations and quality control working solutions with 4 concentrations. The stock solutions and working solutions of the internal standard are prepared in the same manner, and the concentration of the internal standard working solution is consistent with that of the fifth concentration of the calibration working solution. All solutions are stored at -80℃ for later use.

[0108] Preparation of calibrators and quality control samples: Add 10 μL of internal standard working solution and 10 μL of calibrator or quality control working solution of the corresponding concentration to an empty centrifuge tube, and then add 90 μL of blank serum; for actual sample analysis, add 10 μL of internal standard working solution to an empty centrifuge tube, then add 100 μL of serum sample, mix thoroughly, and then process according to the same pretreatment steps.

[0109] Liquid-liquid extraction method for extracting steroid hormones from serum This application employs liquid-liquid extraction to extract the target analyte from serum. After prior optimization, a 1:1 mixture of tert-butyl methyl ether and ethyl acetate was selected as the extraction solvent, with a serum-to-solvent volume ratio of 1:5 yielding the optimal extraction efficiency. Specific procedures: 100 μL of the serum to be tested was added to 500 μL of the above extraction solvent. The mixture was vortexed for 5 min, centrifuged at 14500 rpm for 5 min, and 450 μL of the supernatant was transferred to a new centrifuge tube. The solvent was then evaporated in a vacuum drying oven at 40℃ for later use.

[0110] Oxime reactions of steroid hormones Weigh an appropriate amount of quaternary ammonium oxide and dissolve it in 50% methanol solution to prepare a 0.2 mol / L quaternary ammonium oxide stock solution. Dilute the stock solution with pure methanol and add an appropriate amount of formic acid to prepare a 0.1 mol / L quaternary ammonium oxide working solution containing 2-8% formic acid. Both the stock solution and the working solution should be stored at -80℃ for later use.

[0111] The dried residue containing the target analyte, obtained by liquid-liquid extraction, was added to a centrifuge tube containing 100 μL of quaternary ammonium oxide working solution, and the oxime reaction was carried out at 40 °C for 75 min. (See the schematic diagram of the reaction.) Figure 2 B. After the derivatization reaction is complete, centrifuge at 14500 rpm for 5 min, and take 80 μL of the supernatant for analysis.

[0112] Instruments and analytical conditions Steroid hormones were detected using a reverse-flushing transfer two-dimensional liquid chromatography-tandem mass spectrometry system, which consisted of a Demeter 40 AC HPLC and a Demeter DMT 9600 mass spectrometer equipped with an electrospray ionization source.

[0113] One-dimensional chromatography columns were Waters C18 columns (100 × 4.6 mm, 5 μm; Waters Corporation, Milford, USA) for matrix purification and analyte enrichment; two-dimensional chromatography columns were KF5-W columns (100 × 3.0 mm, 2.6 μm; Demeter Corporation, Changsha, Hunan, China), with a stationary phase of adamantyl and phenylhexyl mixture in a 4:1 volume ratio for analyte separation. Both columns were set to a temperature of 45 °C. Mobile phases consisted of 10 mM ammonium formate aqueous buffer (mobile phase A) and acetonitrile (mobile phase B), both containing 0.1% (v / v) formic acid, at a flow rate of 0.6 mL / min.

[0114] The gradient elution program was as follows: the initial proportion of mobile phase B was 15%; from 0 to 10 min, the proportion of mobile phase B increased to 28%; from 10 to 10.5 min, the proportion of mobile phase B increased to 30%; from 10.5 to 11.5 min, the proportion of mobile phase B remained at 30%; from 11.5 to 16 min, the proportion of mobile phase B increased to 40%; from 16 to 16.1 min, the proportion of mobile phase B increased to 100%, and the column was washed; from 16.1 to 19 min, the proportion of mobile phase B remained at 100%; and from 19.1 min, the proportion of mobile phase B decreased to 15% and was maintained until 21 min to equilibrate the column. Additionally, an auxiliary pump was started from 0.1 to 0.7 min to deliver mobile phase A at a flow rate of 0.6 mL / min.

[0115] During elution, from 0 to 1.5 min, the mobile phase passes forward through the one-dimensional column, enriching the analyte on the column while eluting most impurities. From 1.5 to 4.75 min, a switching valve performs a backwash on the one-dimensional column, eluting the enriched analyte to the two-dimensional column. At 4.75 min, the backwash transfer is complete, the one-dimensional column is disconnected, and the analyte is separated on the two-dimensional column, simultaneously initiating mass spectrometry detection. All derivatized steroid hormones were detected in positive ion mode, with an ion source interface temperature of 220℃, a heating block temperature of 280℃, and a desolvation tube temperature of 250℃. Quantification was performed using multiple reaction monitoring (MRM). The ion pairs and optimized collision energies for each compound are shown in [reference needed]. Figure 3 .

[0116] Methodological Validation This application validates the established method in accordance with the principles of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use M10 Bioanalytical Method Validation and Study Sample Analysis (2022) and the Chinese Expert Consensus on Quality Assurance of Liquid Chromatography-Tandem Mass Spectrometry Detection of Steroid Hormones in Serum / Plasma (2024).

[0117] This application uses spiked calibrators to prepare a standard curve for steroid hormones. The quantitative range is determined based on reference intervals for healthy individuals and patients with polycystic ovary syndrome reported in previous literature, as well as relevant data from the Mayo Clinic website. A 1 / x metric is used. 2 Linear regression analysis was performed using a weighted method.

[0118] Method specificity and selectivity assessment: Six serum samples from different sources were used to prepare double-blank samples, single-blank samples, and limit-of-quantitation (LOQ) samples. Double-blank samples consisted of only blank serum; single-blank samples consisted of blank serum with only an internal standard added; and LQ samples consisted of blank serum with both the LQ working solution and the internal standard added. The requirement was that in double-blank samples, the response value of each internal standard should not exceed 5% of the corresponding response value in the single-blank sample, and the response value of each analyte should not exceed 20% of the corresponding response value in the LQ sample.

[0119] Intra-assay accuracy and precision assessment: In the same analysis, prepare a limit-of-quantitation (LOQ) sample and four quality control samples (QC1~QC4) at four different concentrations, with six replicates for each concentration. Inter-assay accuracy and precision assessment: Prepare samples at the above concentrations for testing in three independent analyses. Intra-assay and inter-assay accuracy are expressed as relative error (RE%), calculated using the formula: RE% = [(Measured value - Nominal value) / Nominal value] × 100%. Intra-assay and inter-assay precision are expressed as relative standard deviation (RSD%), calculated using the formula: RSD% = (Standard deviation / Average value) × 100%. Acceptable standards for accuracy and precision: The relative error and relative standard deviation for the LQ sample should not exceed 20%, and for the remaining quality control samples, they should not exceed 15%.

[0120] Matrix effect assessment: At least six blank matrices from different sources were used, and assays were performed at two concentration levels, QC1 and QC4. For each analyte and internal standard, the matrix factor (MF) for each matrix was calculated using the formula: MF = Peak area with matrix / Peak area without matrix. The normalized matrix factor (ISMF) for the internal standard was then calculated using the formula: ISMF = Analyte matrix factor / Internal standard matrix factor. The relative standard deviation of the normalized matrix factor for the internal standard across different matrices should not exceed 15%.

[0121] Extraction recovery rate (REC%) assessment: In the same analysis, detection was performed at four concentration levels from QC1 to QC4, with four replicates prepared for each concentration. First, the analyte working solution was added to the blank matrix, extracted using standard methods, and then an internal standard was added to obtain the peak area ratio of the analyte to the internal standard (R1). Second, the blank matrix was extracted first, and then the same concentrations of analyte working solution and internal standard were added to the extracted sample to obtain the peak area ratio (R2). The extraction recovery rate was calculated using the formula: REC% = (average of R1 / average of R2) × 100%. The recovery rate was required to remain consistent throughout the calibration range, without a significant concentration-dependent trend.

[0122] Stability assessment: The stability of samples under different storage and processing conditions was examined at two concentration levels, QC1 and QC4. A standard curve of freshly prepared calibrators was used for quantitative analysis of the stability test samples. Refrigerated stability: The stability of samples at 4°C for different time periods was examined. This study tested the stability after 4 hours, 12 hours, and 24 hours at 4°C to determine the longest stable time. Freeze-thaw stability: The stability of samples after multiple freeze-thaw cycles was examined. Samples were frozen at -80°C for at least 12 hours between freeze-thaw cycles. This study examined the stability after three freeze-thaw cycles. Post-preparation stability: The stability of extracted and derivatized samples at the autosampler temperature was examined. The autosampler temperature was set to 4°C in this study, therefore the stability of samples after 12 hours and 24 hours at 4°C was examined. Long-term stability: The stability of samples during long-term storage in a low-temperature freezer was examined. This study examined the stability after 30 days of storage at -80°C.

[0123] Dilution reliability assessment: Prepare quality control samples at a concentration 5 times the upper limit of quantitation, and perform a 10-fold dilution using blank serum. Prepare 6 parallel samples and perform the analysis in the same session. The relative error of the diluted samples should not exceed ±15%, and the relative standard deviation should not exceed 15%.

[0124] Clinical sample analysis The validated method was applied to the analysis of serum samples from patients with both polycystic ovary syndrome (PCOS) and non-PCOS who visited the Reproductive Medicine Center of Peking University Third Hospital. All participants were ≥18 years of age. Given that anti-Müllerian hormone (AMH) is a recognized biomarker for PCOS, participants were divided into two groups based on their AMH levels and clinical characteristics: a PCOS group (AMH ≥10 ng / mL) and a non-PCOS group (AMH <1 ng / mL). Participants with other endocrine disorders were excluded. Fasting venous blood was collected from the participants on the morning of days 2-4 of their menstrual cycle. After blood clotting, the blood was centrifuged at 3000×g for 15 minutes, and the pale yellow supernatant was carefully transferred to sterile centrifuge tubes and stored at -80℃ for later use. This study was approved by the Medical Ethics Committee of Peking University Third Hospital (Approval No.: 2025-295-01).

[0125] Statistical analysis Statistical analysis was performed using SPSS 26.0 software. Normally distributed data were expressed as mean ± standard deviation (x ± SD), and t-tests were used for comparisons between groups. Non-normally distributed data were analyzed using non-parametric tests. A p-value < 0.05 was considered statistically significant. GraphPad Prism 8.0 software was used to create graphs and tables.

[0126] Results and Discussion Method establishment Our previous research team established a detection method based on high-performance liquid chromatography-differential mobility spectrometry / tandem mass spectrometry (HPLC-DMS / MS / MS) capable of detecting eight underivatized androgens. However, this method requires the addition of a differential mobility spectrometry module to the HPLC-DMS system, resulting in poor versatility; furthermore, the mass spectrometric response signal of underivatized androgens is weak, and the detection sensitivity cannot meet the expected requirements. This application addresses the issue from the perspective of derivatization to improve the detection sensitivity of the analytes. Quaternary ammonium oxyamine derivatizing reagents were first applied by Sta-Winstock et al. to the derivatization detection of testosterone, reducing the limit of quantification of testosterone to the pg / mL level without requiring stringent derivatization conditions. Quaternary ammonium oxyamines can undergo oxime reactions with ketone or carbonyl groups in compounds (…). Figure 2 (As shown in B), this significantly enhances the detection sensitivity of the analytes in electrospray tandem mass spectrometry. All 12 steroid hormones detected in this application contain one or two ketone carbonyl groups, and theoretically, all can undergo derivatization reactions with quaternary ammonium oxides. This application screened the parent ions and daughter ions of each analyte after derivatization, and the results are as follows: Figure 3As shown, most analytes exhibited two pairs of ion transitions, with the stronger pair used as the quantitative ion pair and the weaker pair as the qualitative ion pair. Some analytes only exhibited one pair of ion transitions, which were directly used as the quantitative ion pair. For the internal standards of each analyte, only one pair of ions was selected as the reference ion. Among these, progesterone, 17-hydroxyprogesterone, 11β-hydroxyandrostenedione, androstenedione, and 11-ketoandrostenedione all contain two derivatization sites, and the derivatized products carry two charges; therefore, the mass-to-charge ratio of their parent ions is half of the exact molecular weight. The optimized collision energy results for each ion pair are also shown in [the original text]. Figure 3 After derivatization, various steroid hormones can form multiple isomers such as cis and trans. Therefore, it is necessary to further explore the separation effect of derivatized hormones under different chromatographic conditions.

[0127] First, under suitable mobile phase conditions, this application compared the separation performance of derivatized steroid hormone mixtures on EC-C18 columns (100 × 3.0 mm, 2.7 μm, Agilent Poroshell) and adamantyl alkyl columns (100 × 3.0 mm, 3.0 μm, Demeter). This application focuses on four isomers of compounds that are difficult to separate: 17-hydroxyprogesterone-quaternary ammonium oxide, 11-ketoandrostenedione-quaternary ammonium oxide, androstenedione-quaternary ammonium oxide, and progesterone-quaternary ammonium oxide. The separation results are shown in [Figure number missing]. Figure 4 . Figure 4 A and Figure 4 B represents the separation results of the four compounds using the two columns mentioned above. The results show that neither column can achieve good separation of the four compounds, especially the 17-hydroxyprogesterone-quaternary ammonium oxychloride. Considering that the rigid cage structure of the adamantyl column may be sensitive to the shape and planarity of the steroid nucleus, and has limited selectivity for isomers with similar polarity; while the phenylhexyl column can selectively retain polycyclic aromatic hydrocarbons through π-π interactions, this application introduces an appropriate proportion of phenylhexyl into the adamantyl stationary phase to improve the separation ability of analytes with similar polarity. Based on this, this application uses adamantyl and phenylhexyl as packing material. Furthermore, this application screened several columns with different adamantyl to phenylhexyl volume ratios, and finally determined to use a column with a volume ratio of 4:1 (named KF5-W column, 100 × 3.0 mm, 2.6 μm, Demeter). Through systematic screening, the mobile phase system was finally determined to be mobile phase A (a 10 mmol / L ammonium formate aqueous solution containing 0.1% (V / V) formic acid) and mobile phase B (an acetonitrile solution containing 0.1% (V / V) formic acid).

[0128] The gradient elution process is as follows (all proportions mentioned in the elution process are volume proportions).

[0129] The initial proportion of mobile phase B is 15%. Within 0-10 minutes, the proportion of mobile phase B increased to 28%; After 10-10.5 minutes, the proportion of mobile phase B increases to 30%. 10.5-11.5 min, maintain the proportion of mobile phase B at 30%; 11.5-16 min, the proportion of mobile phase B increases to 40%; 16-16.1 min, the proportion of mobile phase B increases to 100%; 16.1–19 min, the proportion of mobile phase B is maintained at 100%; At 19.1 min, the proportion of mobile phase B was reduced to 15% and maintained for 21 min to equilibrate the column.

[0130] The samples were separated using a KF5-W column and an optimized elution protocol, and the results are as follows: Figure 4 As shown in C, the four isomers that were previously difficult to separate have all been successfully separated, proving that the mixed stationary phase strategy adopted in this application has a good separation effect.

[0131] After solving the separation problem, this application introduces a backflushing transfer two-dimensional liquid chromatography technique, further improving detection sensitivity by increasing the injection volume. The processed sample is first enriched and impurities removed on a one-dimensional column, then backflushed and transferred to a two-dimensional column for separation. During the study, the total time and time window of the entire backflushing transfer process need to be determined, while ensuring that the analytes do not elute from the two-dimensional column before the backflushing transfer on the one-dimensional column is complete. The optimized mobile phase gradient conditions (the aforementioned elution scheme) when only the two-dimensional column was connected initially fully considered this requirement, ensuring that all analytes eluted after 7 minutes, allowing sufficient time for backflushing transfer. Subsequently, only the one-dimensional column was connected to the mass spectrometer, and the time window for backflushing transfer was determined. At this time, the mobile phase gradient must be consistent with the optimized two-dimensional column gradient and cannot be arbitrarily changed. Figure 5 As shown, the one-dimensional chromatographic column used in this application is a C18 column (100×4.6mm, 5μm, Waters). This column has a larger inner diameter, larger packing particle size, and longer column length, making it more suitable for large-volume injections and less prone to overload. This study also tested other shorter and finer chromatographic columns, but when these were connected in series with a two-dimensional column, peak splitting still occurred for some analytes during large-volume injections, indicating column overload.

[0132] From 0 to 1.5 min, the mobile phase flows forward through the one-dimensional column at a flow rate of 0.6 mL / min to remove most of the highly polar impurities. To enrich the analytes at the front end of the one-dimensional column and facilitate complete transfer to the two-dimensional column during backwashing, the instrument's auxiliary pump is started from 0.1 to 0.7 min to deliver 100% mobile phase A at a flow rate of 0.6 mL / min. This step adjusts the retention behavior of the analytes and further removes impurities. From 1.5 to 4.75 min, the flow direction is changed by switching the valve to backwash the one-dimensional column, transferring the analytes to the mass spectrometer. The results show that all analytes entered the mass spectrometer and generated response signals before 4.75 min, indicating that the transfer was complete.

[0133] The timing and duration of backwashing and transfer are crucial parameters. Starting backwashing too early will result in insufficient impurity removal, while starting too late may cause the enriched analyte to be flushed out of the one-dimensional column, preventing transfer and potentially affecting the total detection time. The total backwashing and transfer time needs to be controlled to ensure transfer is completed within a short time window. A longer time window may cause some substances flushed into the two-dimensional column to be flushed out before the backwashing ends, preventing them from entering the detector and thus affecting the detection results. Therefore, from... Figure 6 It can be seen that setting the backwash transfer time window to 1.5~4.75 min is more appropriate.

[0134] After optimizing the conditions of the one-dimensional and two-dimensional chromatographic columns respectively, this application connects the two columns in series to establish a two-dimensional liquid chromatography-tandem mass spectrometry detection method based on reverse rinsing transfer. Figure 7 As shown in the results, the detection method of this application can achieve good separation of 12 derivatized steroid hormones. Even when the injection volume is increased to 40 μL, all chromatographic peaks still maintain good peak shape. The sensitivity of the detection method of this application is significantly improved, which can meet the requirements of accurate quantitative analysis.

[0135] Derivatization condition optimization Based on the previously optimized sample pretreatment process, steroid hormones were extracted from serum using liquid-liquid extraction. After extraction, the solvent in the centrifuge tubes was evaporated using a vacuum dryer, followed by the addition of a derivatization reagent containing quaternary ammonium oxide for oxime reaction. To determine the optimal oxime reaction conditions, this application employed a single-factor variable method to systematically optimize the dosage of quaternary ammonium oxide, the volume fraction of formic acid, the reaction temperature, and the reaction time. Figure 8 As shown, the optimal derivatization conditions were finally determined to be 10 μmol quaternary ammonium oxychloride (100 µL, 0.1 mol / L), 8% (V / V) formic acid, and reaction at 40℃ for 75 min.

[0136] Methodological Validation Linearity, precision, accuracy and specificity This application uses eight concentration levels to prepare a standard curve, where the first concentration is the lower limit of quantitation and the eighth concentration is the upper limit of quantitation, using 1 / x 2 Weighted regression analysis was performed. The lower limit of quantitation (LOQ) for each analyte was 0.005–0.1 ng / mL, and the upper limit of quantitation (UPQ) was 200 times its LQ. The quantitation ranges for each analyte are shown below. Figure 9 As shown in the figure, the data indicate that all analytes exhibit good linearity within their respective quantitative ranges, with correlation coefficients (r) of [value missing]. 2 All are ≥0.99.

[0137] The intra- and inter-batch accuracy and precision of the method were evaluated at the limit of quantitation and five concentration levels (QC1-QC4). The results showed that the accuracy (relative error) of all analytes was within ±15%, and the intra- and inter-batch precision (relative standard deviation) did not exceed 15%.

[0138] The method specificity was evaluated using human serum samples from six different individuals. The results showed that in the double blank samples treated with activated carbon, the response values ​​of each analyte did not exceed 20% of those in the limit of quantitation samples, and the response values ​​of the internal standard did not exceed 5% of those in the single blank samples.

[0139] Matrix effect and extraction recovery Six serum samples from different sources were used to assess the matrix effect at two concentration levels, QC1 and QC4. The results are shown in [Figure number missing]. Figure 10-14 The relative standard deviations of the matrix factor normalized by the internal standard for each analyte at both concentration levels were all less than 15%, indicating that the matrix effect had no significant impact on the accurate quantification of the analytes.

[0140] The extraction recoveries of each analyte were evaluated at four concentration levels, QC1 to QC4, with four replicates prepared for each concentration. Figure 10 The results showed that the extraction recoveries of each analyte remained consistent at different concentrations, with relative standard deviations all less than 15%, and no significant concentration-dependent changes.

[0141] Stability and Dilution Reliability At two concentration levels, QC1 and QC4, the stability of the sample under different conditions was examined. A sample was considered stable under these conditions if the accuracy (relative error) was within ±15% and the precision (relative standard deviation) did not exceed 15%. Figure 15As shown. Refrigerated stability: The stability of samples after being stored at 4°C for 4 hours, 12 hours, and 24 hours was examined. The results showed that, except for 11-hydroxytestosterone which was stable for only 4 hours at 4°C and progesterone which was stable for only 12 hours, the other analytes remained stable after 24 hours at 4°C. Freeze-thaw stability: Serum samples containing the analytes were subjected to three freeze-thaw cycles at -80°C. The results showed that, except for 11-hydroxytestosterone and progesterone which could only withstand two freeze-thaw cycles, the other compounds remained stable after three freeze-thaw cycles. Post-preparation stability: The stability of samples after pretreatment and derivatization was examined after being stored at 4°C for 4 hours, 12 hours, and 24 hours. The results were consistent with refrigerated stability; except for 11-hydroxytestosterone which was stable for only 4 hours and progesterone for only 12 hours, the other analytes remained stable for 24 hours. Long-term stability: The stability of samples stored at -80°C for 30 days was examined. The results showed that all analytes remained stable. The above results indicate that 11-hydroxytestosterone and progesterone are less stable than other analytes. Therefore, after blood collection, serum extraction should be completed within 4 hours if possible, followed by storage at -80°C for no more than 30 days. The analysis of each batch of samples should be completed within 4 hours, with no more than 2 freeze-thaw cycles.

[0142] When the concentration of the analyte in the sample exceeds the upper limit of quantification, the sample needs to be appropriately diluted. To verify the reliability of the dilution operation, this application first prepared a serum sample containing the analyte at a concentration 5 times the upper limit of quantification. After diluting the sample 10-fold with blank serum, the accuracy of quantification of the diluted analyte was evaluated. The results are shown in Table 3. At the two concentration levels of QC1 and QC4, the accuracy (relative error) of all parallel samples was within ±15%, and the precision (relative standard deviation) was less than 15%, indicating that all analytes can be accurately quantified after 10-fold dilution.

[0143] Clinical Sample Application Based on the aforementioned inclusion and exclusion criteria, and after age matching, this application included 96 non-polycystic ovary syndrome (PCOS) participants and 100 PCOS participants, whose baseline clinical characteristics are as follows: Figure 16 As shown, the luteinizing hormone (LH) / follicle-stimulating hormone (FSH) ratio and progesterone levels in the polycystic ovary syndrome (PCOS) group were significantly higher than those in the non-PCOS group, consistent with previously reported characteristics of the PCOS population. After comprehensive methodological validation, the established reverse-flushing transfer two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS / MS) method was used to detect 12 steroid hormones in actual serum samples. The results are detailed in [link to results]. Figure 17 and Figure 18 . Figure 19The chromatograms shown are representative of some low-concentration actual serum samples. The results indicate that there is almost no interference from other substances for each analyte, ensuring the reliability of the quantitative results. It is worth noting that this study set a relatively low limit of quantification (0.05 ng / mL) for progesterone, but the progesterone concentration detected in some patients was still below this limit; compared with the results of clinical immunoassay, the progesterone concentration detected by this method was significantly lower. A search of the public chemistry database of the National Center for Biotechnology Information (NCBI) revealed that progesterone is photodegradable. Although light-protection measures were taken during the methodological validation process in this study, complete light protection is difficult to achieve during clinical sample collection and processing. Progesterone in low-concentration samples may undergo partial degradation due to prolonged light exposure, thus affecting the accuracy of quantification. Therefore, this study excluded progesterone detection data from subsequent statistical analysis and used the results of clinical immunoassay as a reference. The concentrations of the remaining 11 hormones were all within the quantitative range of this method. Except for 11-hydroxytestosterone and 11-ketotestosterone, which showed no statistically significant difference between the two groups, the levels of the other nine steroid hormones in the polycystic ovary syndrome (PCOS) group were significantly higher than those in the non-PCOS group. These results not only provide strong evidence for further research into the differences in sex hormone profiles between PCOS and non-PCOS individuals, but also fully demonstrate the applicability of this detection method in steroid hormone detection in both PCOS and non-PCOS populations.

[0144] Conclusions and Discussion This application successfully established a reverse-flushing transfer two-dimensional liquid chromatography-tandem mass spectrometry method, capable of simultaneously determining four progestins and eight androgens in the serum of individuals with and without polycystic ovary syndrome (PCOS). This method not only achieves efficient separation of multiple steroid hormones but also increases the injection volume to 40 μL without sample pre-concentration. Through system optimization and extensive experimental validation, this method effectively removes matrix interference and significantly increases the amount of target analyte entering the detector, thereby greatly improving detection sensitivity. However, it must be noted that due to the lack of strict light protection measures during clinical sample collection, progesterone in some samples underwent significant degradation, making accurate statistical analysis of the detection data impossible. This issue needs to be addressed and resolved in future research.

[0145] In recent years, numerous studies have focused on developing liquid chromatography-tandem mass spectrometry (LC-MS / MS) methods for the quantitative detection of steroid hormones. This study summarizes relevant research from the past five years. Figure 20(As shown). Compared to most reported methods, our method requires only a small amount of clinical samples and eliminates the need for pre-concentration steps (as described in our previous study CN2025117464533), while achieving superior detection sensitivity. It is particularly noteworthy that current methods for detecting 11-oxidized androgens are still relatively limited, with few studies measuring 11-oxidized androgen levels in patients with polycystic ovary syndrome. Therefore, our work fills a gap in this field to some extent. This application compares our established method with two excellent steroid hormone detection studies published in recent years: Luke Cordova et al. established an automated solid-phase extraction-liquid chromatography-tandem mass spectrometry method that can detect 16 steroid hormones under non-derivative conditions with extremely high sensitivity, almost reaching the current limits of sex hormone detection. This study used 175 μL of serum. After automated solid-phase extraction, all steroid hormones in the sample could be separated and detected by the chromatographic system, resulting in an extremely low limit of quantitation. However, this method relies on an expensive automated solid-phase extraction-liquid chromatography-tandem mass spectrometry platform, which is only available in a few testing institutions, limiting its widespread application. Adrianson et al. established a two-dimensional liquid chromatography-tandem mass spectrometry method that can detect four classic androgens and four 11-oxidized androgens, and successfully applied it to establish reference ranges for relevant hormones in children's serum. This method also has high sensitivity and is suitable for populations with low hormone levels, such as children. However, this study used two independent detection methods for different androgen subgroups, significantly increasing the complexity and time consumption of the analysis. In addition, after analyzing the chromatographic conditions of this method, we found that the column temperature was set to 60℃ and the mobile phase flow rate changed frequently, which placed high demands on the durability of the chromatographic column and the stability of the system. In contrast, the method in this study is based on a more economical detection platform, with mild and stable chromatographic conditions. The column temperature, pressure, and flow rate are all within the conventional range, which significantly improves the detection sensitivity while ensuring the stability of the detection results and the wide applicability of the method.

[0146] This study also yielded significant findings regarding the detection results in individuals with and without polycystic ovary syndrome (PCOS). Previous studies have confirmed elevated levels of classical androgens in PCOS patients, and this study similarly found that PCOS patients had significantly higher levels of dehydroepiandrosterone (DHEA), testosterone, dihydrotestosterone, and androstenedione than the non-PCOS group. Furthermore, this study is the first to report significantly elevated levels of two 11-oxidized androgens (11-hydroxyandrostenedione and 11-ketoandrostenedione) in the serum of PCOS patients, a phenomenon rarely documented in existing literature. While our previous small-sample study observed a slight increase in 11-oxidized androgen levels in the PCOS group, the difference was not statistically significant due to the small sample size. This application, by expanding the sample size, confirms that the difference between the two groups is statistically significant. Regarding progestins, previous studies have focused relatively little on them, and the results have been inconsistent. This application confirms (using clinical immunoassay for progesterone) that the levels of all four progestins in patients with polycystic ovary syndrome (PCOS) were significantly higher than in the non-PCOS group. Our previous study included 35 PCOS patients and 34 non-PCOS participants, measuring three progestins: 17-hydroxyprogesterone, 17-hydroxypregnenolone, and pregnenolone. Although an upward trend in progesterone levels was observed in the PCOS group, the difference was not statistically significant due to the limited sample size. This application, by increasing the sample size by nearly three times, shows a statistically significant difference between the two groups. This indicates that progestins, upstream of androgen synthesis, may also play an important role in the pathophysiology of PCOS, requiring further in-depth analysis of steroid hormone metabolic pathways.

[0147] In summary, the reverse-flushing transfer two-dimensional liquid chromatography-tandem mass spectrometry method established in this study has been successfully applied to the detection of steroid hormones in actual clinical samples, achieving satisfactory results. It has great application potential in future clinical research and diagnostic practice.

[0148] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0149] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for simultaneously detecting multiple steroid hormones in serum, wherein, The method includes: Hormones were extracted from serum using liquid-liquid extraction. A derivatization reagent is added to the extracted hormone to carry out an oxime reaction, wherein the derivatization reagent contains quaternary ammonium oxyamine; The hormone after oxime reaction is injected into a two-dimensional liquid chromatography-tandem mass spectrometry system for detection. The hormone is enriched on a one-dimensional chromatographic column, and then the enriched hormone is backwashed and transferred to a two-dimensional chromatographic column for analysis.

2. The method according to claim 1, wherein, The packing material for the two-dimensional chromatographic column includes adamantyl and / or phenylhexyl groups.

3. The method according to claim 2, wherein, The volume ratio of adamantyl to phenylhexyl in the two-dimensional chromatographic column is 4:

1.

4. The method according to claim 1, wherein, During detection, gradient elution is performed using a mixed mobile phase consisting of mobile phase A and mobile phase B. Mobile phase A is a 10 mM aqueous solution of ammonium formate containing 0.1% (v / v) formic acid, and mobile phase B is acetonitrile containing 0.1% (v / v) formic acid. During gradient elution, the volume percentage of mobile phase B in the mixed mobile phase gradually increases from the initial percentage and then returns to the initial percentage. During gradient elution, the elution temperature was 45℃, the flow rate was 0.6 mL / min, and the elution time was 21 min.

5. The method according to claim 4, wherein, When the hormone is enriched on a one-dimensional chromatographic column, the mobile phase A is also delivered to the one-dimensional chromatographic column via an auxiliary pump.

6. The method according to claim 1, wherein, The total time for the reverse flushing transfer is 0.1-5 min.

7. The method according to claim 1, wherein, The derivatizing reagent also includes methanol, water and formic acid, wherein the concentration of formic acid is 2-15% (V / V).

8. The method according to claim 1, wherein, The reaction temperature for oximeization is 30-50℃, and the reaction time is 60-90 min.

9. The method according to claim 3, wherein, The one-dimensional chromatographic column is a C18 column with dimensions of 100×4.6mm and 5μm; the two-dimensional chromatographic column has dimensions of 100×3mm and 2.6μm.

10. The method according to claim 1, wherein, The steroid hormones mentioned are selected from testosterone, dehydroepiandrosterone, dihydrotestosterone, androstenedione, 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketotestosterone, 11-ketoandrostenedione, progesterone, 17-hydroxyprogesterone, pregnenolone, and 17-hydroxypregnenolone.

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