Method for detecting various free androgens in serum

Abstract

The invention relates to a method for detecting various free androgens in serum, which comprises the following steps: carrying out ultrafiltration treatment on a sample to obtain an ultrafiltration solution; magnetic beads are added into the ultrafiltration solution, and androgen is extracted through magnetic solid-phase extraction; adding a derivatization reagent containing quaternary ammonium oxygen amine into the extracted androgen, and carrying out oximation reaction; and injecting the solution after oximation reaction into a high performance liquid chromatography-tandem mass spectrometry system for detection. The method provided by the invention has excellent specificity and sensitivity, the linear fitting correlation coefficient (r) is greater than 0.99, the recovery rate of the extract is higher than 95.1%, the precision and accuracy measured by relative errors and variable coefficients both meet the regulations of the clinical and laboratory standard association (CLSI), and a stability test proves that under typical clinical treatment conditions, the analyte loss is extremely small.

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 the simultaneous detection of multiple free androgens in serum based on ultrafiltration-high performance liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Traditional research on androgens has primarily focused on testosterone (T), androstenedione (A4), dehydroepiandrosterone (DHEA), and dihydrotestosterone (DHT). In recent years, novel androgens, specifically 11-oxyandrogens, have attracted increasing attention. These hormones include 11β-hydroxyandrostenedione (11OHA4), 11β-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), and 11-ketodihydrotestosterone (11KDHT), all characterized by a hydroxyl or carbonyl group at the 11th carbon atom. Studies have shown that 11-oxyandrogens are associated with various pathological conditions, such as congenital adrenal hyperplasia (CAH), polycystic ovary syndrome (PCOS), and castration-resistant prostate cancer (CRPC).

[0003] In the circulatory system, androgens exist primarily in three forms: protein-bound, bound, and free. Most hormones in the body are bound to carrier proteins, mainly sex hormone-binding globulin (SHBG) and human serum albumin (HSA). Free androgens account for less than 3% of the total androgen concentration, but they are the biologically active form—they can easily diffuse into target cells and bind directly to androgen receptors, playing a crucial role in human growth, development, and reproductive health. Given their vital role in the body, free androgens have become important biomarkers for various diseases such as hypogonadism and hyperandrogenism.

[0004] However, direct detection of free androgens faces significant challenges due to their low concentration and strong affinity for binding proteins. Most studies rely on mass action equations to estimate free testosterone levels. These calculations depend on the concentrations of sex hormone-binding globulin (SHBG), total testosterone, or albumin, which can lead to inconsistencies in results. Currently available direct detection techniques include balanced dialysis, centrifugal ultrafiltration, steady-state gel filtration, flow dialysis, and direct tracer analog immunoassay.

[0005] Among these methods, centrifugal ultrafiltration is widely favored due to its good reproducibility, ease of operation, and minimal interference from the sample matrix. This technique utilizes a filter membrane with a specific molecular weight cutoff (MWCO) to effectively separate free androgens from protein-bound components such as SHBG (molecular weight 94 kDa) and albumin (molecular weight 66.5 kDa).

[0006] The inventors had previously developed a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method for determining free testosterone and androstenedione in serum, laying a solid foundation for subsequent research. Summary of the Invention

[0007] To further improve the sensitivity and reproducibility of androgen detection and analysis, and to optimize the method for androgen detection, this application combines ultrafiltration with liquid chromatography-tandem mass spectrometry to provide a detection method that is highly sensitive, reproducible, simple and efficient, and very suitable for clinical and research scenarios. This method can simultaneously quantify multiple free androgens in serum, opening up new directions for androgen-related pathophysiological research (especially androgen excess or deficiency-related diseases).

[0008] 1. A method for detecting multiple free androgens in serum, wherein the method comprises: The sample was subjected to ultrafiltration to obtain an ultrafiltration solution; Magnetic beads were added to the ultrafiltration solution, and androgens were extracted by magnetic solid-phase extraction. A derivatization reagent containing quaternary ammonium oxide was added to the extracted androgens to carry out an oxime reaction; The oxime-treated solution was injected into a high-performance liquid chromatography-tandem mass spectrometry system for detection.

[0009] 2. The method according to claim 1, wherein the ultrafiltration membrane is pretreated with an aminosilane coupling agent before ultrafiltration, preferably a monoaminosilane coupling agent, more preferably 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane or 4-aminobutyltriethoxysilane.

[0010] 3. The method according to item 2, wherein the pretreatment comprises soaking the ultrafiltration membrane in an aminosilane coupling agent solution for 1-5 hours, wherein the volume ratio of the coupling agent to the ultrafiltration solution is (1-10):100.

[0011] 4. The method according to item 1, wherein the magnetic bead is a silicon-based steroid magnetic bead.

[0012] 5. The method according to item 1, wherein 0.02-2 mg of magnetic beads, preferably 0.1-1 mg, is added to every 600 μL of the ultrafiltration solution.

[0013] 6. The method according to item 1, wherein the adsorption time of magnetic solid phase extraction is 1-60 min, preferably 1-15 min.

[0014] 7. The method according to claim 1, wherein 1-20 μmol of quaternary ammonium oxygenase derivatizing reagent, preferably 2-8 μmol of quaternary ammonium oxygenase derivatizing reagent, is added to the androgen extracted from every 600 μL of the ultrafiltration solution to carry out an oxime reaction.

[0015] 8. The method according to claim 1, wherein the reaction substrate for the oxime reaction is an aqueous methanol solution with a methanol concentration of 80% (V / V); and the catalyst for the oxime reaction is an aqueous formic acid solution with a formic acid concentration of 8% (V / V).

[0016] 9. The method according to item 1, wherein the reaction temperature of the oxime reaction is 20-60°C, preferably 40-50°C; and the reaction time of the oxime reaction is 1.5-3 hours.

[0017] 10. The method according to item 1, wherein the chromatographic column used in the high-performance liquid chromatography is an ACQUITY UPLCBEH C18 column with dimensions of 2.1 × 50 mm and a diameter of 1.7 μm; or Gradient elution is performed in high-performance liquid chromatography 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, and mobile phase B is acetonitrile containing 0.1% (v / v) formic acid; or The gradient elution conditions were: elution temperature 45℃, flow rate 0.32 mL / min, and elution time 15 min; or 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.

[0018] This application combines ultrafiltration with liquid chromatography-tandem mass spectrometry (LC-MS / MS) to provide a method for the quantitative detection of multiple free androgens in serum. This method effectively extracts free androgens through ultrafiltration, followed by magnetic solid-phase extraction (MSPE) to reduce matrix interference. Subsequently, QAO reagent is introduced to induce oxime elution, allowing free androgens to elute from the magnetic beads under mild conditions, reducing interference and improving selectivity. Because the ultrafiltrate has a lower viscosity than serum, the mass transfer efficiency is high, resulting in high adsorption efficiency. The recovery rate of the method described in this application is significantly improved compared to serum matrix.

[0019] This application optimizes each step in ultrafiltration, magnetic solid-phase extraction, oxime reaction, and high-performance liquid chromatography-tandem mass spectrometry analysis, developing a novel, reliable, and efficient method for the simultaneous detection of nine free androgens in serum. This method exhibits excellent specificity and sensitivity, providing important insights into the significance of free androgens in health and disease. In the future, this method is expected to improve the accuracy of disease diagnosis, aid in treatment monitoring, and deepen our understanding of androgen-related diseases. Attached Figure Description

[0020] Figure 1 It includes the calibration range, limit of quantitation, and quality control standards for the target analyte and internal standard.

[0021] Figure 2 These are the mass spectrometry parameters of the analyte and internal standard in multiple reaction monitoring mode.

[0022] Figure 3 The regenerated cellulose membrane is pretreated using various pretreatment methods to achieve different non-specific adsorption rates of androgens.

[0023] Figure 4 This describes the extraction effect of different magnetic beads on androgens in ultrafiltration solutions of the same concentration.

[0024] Figure 5 This relates to the effect of the amount of magnetic beads used on the intensity of androgen signals.

[0025] Figure 6 This relates to the effect of adsorption time on the intensity of androgen signals.

[0026] Figure 7 This relates to the effect of magnetic bead rinsing conditions on sensitivity and matrix interference.

[0027] Figure 8 This relates to the effect of the oxime reaction matrix on the derivatization efficiency.

[0028] Figure 9 This relates to the effect of QAO reagent dosage on androgen signal intensity.

[0029] Figure 10 This relates to the effect of oxime reaction temperature on androgen signal intensity.

[0030] Figure 11 This relates to the effect of oxime reaction time on androgen signal intensity.

[0031] Figure 12 This refers to the effect of the catalyst on the intensity of androgen signals in the oxime reaction.

[0032] Figure 13 These are typical chromatograms of nine androgen-QAO derivatives.

[0033] Figure 14 The specificity was assessed using six ultrafiltration solutions of human serum pretreated with activated charcoal, including representative chromatograms of double blank (DB), single blank (SB), lower limit of quantitation (LLOQ) samples, and real patient samples.

[0034] Figure 15 It represents the intra-day and inter-day precision / accuracy of nine analytes across three analytical batches.

[0035] Figure 16 The matrix effect and recovery rate of the nine analytes are shown.

[0036] Figure 17 It refers to the stability of serum samples before ultrafiltration.

[0037] Figure 18 The results are based on the analysis of the distribution characteristics and statistical properties of nine free androgens in the serum of two groups of subjects using the method described in this application.

[0038] Figure 19 It represents the concentration of free androgens in the four substrates. Detailed Implementation

[0039] Specific embodiments of this application will now be described in more detail with reference to the accompanying drawings. While specific embodiments of this application are shown in the drawings, it should be understood that this application can be implemented in various forms and should not be limited to the embodiments or examples set forth herein. Rather, these embodiments 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.

[0040] 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.

[0041] Quaternary ammonium oxide derivatizing reagents refer to reagents containing aminooxygen groups that can react with steroid carbonyl groups. In this application, "quaternary ammonium oxide" and "QAO" can be used interchangeably.

[0042] The nine free hormones involved in this application include T, DHEA, A4, DHT, 11KT, 11OHT, 11OHA4, 11KA4, and 11KDHT.

[0043] The specific detection method described in this application is as follows.

[0044] Serum samples were diluted and ultrafiltered through a centrifugal filtration unit containing a regenerated cellulose membrane to obtain an ultrafiltration solution. Magnetic beads were then added to the ultrafiltration solution, and free androgens in the ultrafiltration solution were extracted and enriched by magnetic solid-phase extraction (MSPE). Quaternary ammonium oxy (QAO) derivatization reagent was then added to the extracted androgens to carry out an oxime reaction. Finally, the oxime-treated solution was injected into a high-performance liquid chromatography-tandem mass spectrometry system for detection.

[0045] In some implementations, the ultrafiltration membrane is pretreated with an aminosilane coupling agent prior to ultrafiltration.

[0046] Aminosilane coupling agents are a class of organosilicon compounds that contain both amino groups (-NH2, -NH-, etc.) and siloxane groups in their molecules. They can establish chemical bonds between inorganic materials (such as metals, glass, ceramics, and silicon-based magnetic beads) and organic materials (such as resins and polymers), and are widely used in material modification, surface modification, adhesives and other fields.

[0047] In some embodiments, the aminosilane coupling agent is a monoaminosilane coupling agent, specifically, it can be any one of 3-aminopropyltrimethoxysilane (which can be used interchangeably with "KH2881" or "coupling agent KH2881" in this application), 3-aminopropyltriethoxysilane, and 4-aminobutyltriethoxysilane.

[0048] In some embodiments, the pretreatment includes soaking the ultrafiltration membrane in an aminosilane coupling agent solution for 1-5 hours, specifically, soaking for 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours.

[0049] In some embodiments, the pretreatment includes soaking the ultrafiltration membrane in an aminosilane coupling agent solution for 2 hours.

[0050] In some embodiments, the volume ratio of the aminosilane coupling agent solution to the ultrafiltration solution is (1-10):100, specifically, it can be 1:100, 2:100, 3:100, 4:100, 5:100, 6:100, 7:100, 8:100, 9:100, or 10:100.

[0051] In some embodiments, the volume ratio of the aminosilane coupling agent solution to the ultrafiltration solution is 5:100.

[0052] In some embodiments, the pretreatment includes soaking the ultrafiltration membrane in a 3-aminopropyltrimethoxysilane solution for 2 hours, wherein the volume ratio of the 3-aminopropyltrimethoxysilane solution to the ultrafiltration solution is 5:100.

[0053] In some implementations, serum samples are diluted 1:1 (v / v) using phosphate-buffered saline (PBS).

[0054] In some embodiments, the magnetic beads are silicon-based steroid magnetic beads.

[0055] In some embodiments, 0.02-2 mg of magnetic beads are added to every 600 μL of the ultrafiltration solution. Specifically, the amounts can be 0.02 mg, 0.05 mg, 0.08 mg, 0.1 mg, 0.12 mg, 0.15 mg, 0.18 mg, 0.2 mg, 0.22 mg, 0.25 mg, 0.28 mg, 0.3 mg, 0.32 mg, 0.35 mg, 0.38 mg, 0.4 mg, 0.42 mg, 0.45 mg, 0.48 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.16 mg, 1.7 mg, 1.8 mg, 1.9 mg, or 2 mg.

[0056] In some embodiments, 0.1-1 mg of magnetic beads are added to every 600 μL of the ultrafiltration solution.

[0057] In some embodiments, 0.1-0.5 mg of magnetic beads are added to every 600 μL of the ultrafiltration solution.

[0058] In some implementations, 0.5 mg of magnetic beads are added to every 600 μL of the ultrafiltration solution.

[0059] In some implementations, the adsorption time for magnetic solid phase extraction is 1-60 min, specifically 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, and 60 min.

[0060] In some implementations, the adsorption time for magnetic solid-phase extraction is 1-15 min.

[0061] In some implementations, the adsorption time for magnetic solid-phase extraction is 15 min.

[0062] In this application, the oxime reaction can significantly improve the ionization efficiency of androgens in mass spectrometry. This application uses a quaternary ammonium oxygenase derivatizing reagent to perform the oxime reaction. Those skilled in the art should understand that any quaternary ammonium oxygenase derivatizing reagent containing an amino group can achieve this application, regardless of the anion bonded to the amino group, such as QAO·Br, QAO·Cl, etc. in QAO halides, where the Br or Cl ions do not affect the oxime reaction of androgens.

[0063] In some embodiments, 1-20 μmol of quaternary ammonium oxide derivatizing reagent is added to each 600 μL of the ultrafiltration solution to carry out an oxime reaction. Specifically, the values ​​can be 1 μmol, 1.5 μmol, 2 μmol, 2.5 μmol, 3 μmol, 3.5 μmol, 4 μmol, 4.5 μmol, 5 μmol, 5.5 μmol, 6 μmol, 6.5 μmol, 7 μmol, 7.5 μmol, 8 μmol, 8.5 μmol, 9 μmol, 9.5 μmol, 10 μmol, 0.15 μmol, 11 μmol, 11.5 μmol, 12 μmol, 12.5 μmol, 13 μmol, 13.5 μmol, 14 μmol, 14.5 μmol, 15 μmol, 15.5 μmol, 16 μmol, 16.5 μmol, 17 μmol, 17.5 μmol, 18 μmol, 18.5 μmol, 19 μmol, 19.5 μmol, and 20 μmol.

[0064] In some embodiments, 2-8 μmol of quaternary ammonium oxyamine derivatizing reagent is added to each 600 μL of the ultrafiltration solution to carry out an oxime reaction.

[0065] In some embodiments, the reaction substrate for the oxime reaction is an aqueous methanol solution with a methanol concentration of 80% (V / V).

[0066] In some embodiments, the catalyst for the oxime reaction is an aqueous formic acid solution at a concentration of 8% (V / V).

[0067] 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.

[0068] In some embodiments, the oxime reaction is carried out at a temperature of 40-50°C.

[0069] In some embodiments, the oxime reaction is carried out at a temperature of 40°C.

[0070] In some implementations, the reaction time of the oxime reaction is 1.5-3 hours, specifically, it can be 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours.

[0071] In some embodiments, the oxime reaction takes 1.5 hours.

[0072] In some implementations, the HPLC uses a liquid chromatography column with a C18 stationary phase (C18 liquid chromatography column).

[0073] In some implementations, the chromatographic column used is an octadecylsilane-bonded porous silica column.

[0074] 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 columns (2.1×50 mm, 1.7 μm), Poroshell EC-C18 columns (2.1×50 mm, 2.7 μm), or Poroshell EC-C18 columns (2.1×150 mm, 2.7 μm).

[0075] In some implementations, the chromatographic column used is a fluorophenyl column, specifically a Poroshell 120 EC-C18 column, an HSS T3 column, or an HSS PFP column.

[0076] Those skilled in the art will know that any mobile phase that enables the steroid hormones to be tested to achieve adequate separation and good peak shape can be used in the scheme of this application.

[0077] In some implementations, chromatographic separation is performed using an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm). The mixed mobile phase used 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 returning it to the initial percentage.

[0078] In some embodiments, the mobile phase A comprises an aqueous solution of ammonium formate, and the mobile phase B comprises acetonitrile.

[0079] In some embodiments, the concentration of ammonium formate in the mobile phase A is 5-15 mM, such as 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, etc.

[0080] In some preferred embodiments, the concentration of ammonium formate is 10 mM.

[0081] In some embodiments, the mobile phase B further comprises an acid for adjusting the pH value.

[0082] In some embodiments, the acid is formic acid, and the content is 0.1% (v / v).

[0083] In some embodiments, the mobile phase A is an aqueous solution of ammonium formate with a concentration of 10 mM, and the mobile phase B is acetonitrile containing 0.1% (v / v) formic acid.

[0084] 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.

[0085] In some implementations, the gradient elution temperature is 45°C.

[0086] In some implementations, the flow rate of the mobile phase is 0.32 mL / min.

[0087] In some implementations, the elution time is 15 minutes.

[0088] In some implementations, the gradient elution procedure is as follows.

[0089] In the first 0-0.8 minutes, the volume percentage of mobile phase B was 10%. At 1.2 minutes, the volume percentage of mobile phase B increased to 20%; At 5.5 minutes, the volume percentage of mobile phase B increased to 22%. At the 7th minute, the volume percentage of mobile phase B increased to 25%; At 10.5 minutes, the volume percentage of mobile phase B increased to 38%. At the 11th minute, the volume fraction of mobile phase B increased to 95%. From 11.2 to 14 minutes, the volume percentage of mobile phase B was 100%. At 14.2-15 minutes, the volume percentage of mobile phase B was 10%.

[0090] The preferred embodiments of this application have been described in detail above; however, this application is not limited thereto. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application.

[0091] The exemplary embodiments of this application are described below with reference to the accompanying drawings, including various details of the embodiments of this application to aid understanding, and the results demonstrate the technical effects that can generally be achieved by the implementation methods covered by this application.

[0092] Example Material sources used in the embodiments Testosterone (T) was purchased from European Pharmacopoeia; androstenedione (A4) was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China); dehydroepiandrosterone (DHEA) was purchased from Med Chem Express; dihydrotestosterone (DHT) and 11-ketotestosterone (11KT) were purchased from CFWLABs (Walnut, CA, USA); 11β-hydroxytestosterone (11OHT), 11β-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), and 11-ketodihydroandrostosterone (11KDHT) were all purchased from Steraloids (Newport, RI, USA); 11OHA4-d4, T-d3, and DHEA-d6 were purchased from Cambridge Isotope Laboratories Inc. (Andover, MA, USA). (USA). Of the isotope-labeled internal standards, 11OHA4-d4, T-d3, and DHEA-d6 were purchased from Cambridge Isotope Laboratories Inc (Andover, MA, USA), and A4-d7 was purchased from Bepure (Philadelphia, Pennsylvania, USA). HPLC-grade acetonitrile, methanol, and formic acid for high-performance liquid chromatography (HPLC) were purchased from Thermo Fisher Scientific (Fair Lawn, NJ, USA), and ammonium formate was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). (O-(3-trimethylammoniumpropyl)hydroxylamine) bromide (QAO·Br) was purchased from Pharmaron Inc. (Beijing, China). KH2881 reagent was purchased from Jessica Chemical (Hangzhou, China). 5 mg / mL hydrocortisone injection was purchased from Tianjin Jinyao Group Hubei Tianyao Pharmaceutical Co., Ltd. Phosphate-buffered saline (PBS, 1×, 0.01 M, pH 10) was used. 7.2-7.4) Dilute serum samples. Hormone-free human plasma (CMS-S105) was purchased from Shanghai Pufen Biotechnology CO.

[0093] Centrifugal filtration units containing regenerated cellulose membranes (1 mL, molecular weight cutoff 30 kDa) were purchased from Merck Millipore Ltd. (Darmstadt, Germany). Commercial magnetic beads (MSi050 / Steroid MB, MSi050 / Steroid MB-Q, MSi050 / Steroid MB-T (MBT), MSi050 / PLS, MPS10K / PLS, MPS10K / Steroid MB, MPS05K / Steroid MB) were purchased from Jiangsu Province Enriching Biotechnology. Before use, all beads were prepared with isopropanol to a concentration of 50 mg / mL. HLB magnetic beads were purchased from Suzhou Nanwei Life Sciences Technology Co., Ltd., and MGO magnetic beads were purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.

[0094] Clinical sample sources used in the examples This clinical study included 86 infertile women, aged 21-50 years, with a mean age of 33.5 ± 6.5 years. All participants were from Peking University Third Hospital, and the study protocol was approved by the Medical Ethics Committee of Peking University Third Hospital (Approval No.: IRB00006761-M2022236). Blood samples were collected from participants on the morning of days 2-5 of their menstrual cycle. Serum anti-Müllerian hormone (AMH) levels were measured using electrochemiluminescence immunoassay (ECLIA) at the Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology affiliated to Peking University Third Hospital.

[0095] Preparation of calibration standards, quality control samples and solutions A series of stock solutions and working solutions were prepared using methanol as the solvent, and all solutions were stored at -80°C for later use. For specific calibration ranges, limits of quantitation (LLOQ), and quality control (QC) standard concentrations, please refer to [link to relevant documentation]. Figure 1 When preparing calibration standards and quality control samples, a laboratory-made artificial blank ultrafiltration solution was used as the blank matrix; 30 μL of working solution was mixed with 570 μL of artificial blank ultrafiltration solution to obtain the calibration standards.

[0096] Use deionized water ( dThe 5% KH2881 reagent (hereinafter referred to as "5% KH2881 solution") was prepared by diluting the ultrafiltration solution volume of 5% by 20 times. Hydrocortisone, triamcinolone, prednisolone, and dexamethasone were adjusted to a final concentration of 5 μg / mL. Tween-20 was added to 1% low-saturated fatty acid bovine serum albumin (BSA) to a final concentration of 0.05%. The QAO reagent was prepared with methanol to a concentration of 200 mM and diluted before use.

[0097] Ultrafiltration steps Ultrafiltration was performed using a centrifugal filtration unit containing a regenerated cellulose membrane. To reduce non-specific adsorption, this application evaluated various membrane pretreatment methods to screen for the optimal solution, specifically including the following pretreatment methods: (1) Rinse with deionized water; (2) Rinse with 0.1 M sodium hydroxide; (3) Soak in 1% BSA solution for 30 minutes; (4) Soak in 5% KH2881 solution for more than 2 hours.

[0098] (5) First, soak the ultrafiltration tube in 5% KH2881 solution, and then soak it in 5 μg / mL glucocorticoid solution for more than 2 hours, wherein the glucocorticoid is hydrocortisone, triamcinolone, prednisolone or dexamethasone.

[0099] Quality control sample QC4 was prepared using an artificial blank ultrafiltration solution. After ultrafiltration in the pretreated ultrafiltration tube, magnetic solid-phase extraction (MSPE) and in-situ derivatization were performed. A negative control sample QC4 was also included, which underwent MSPE and derivatization directly without ultrafiltration. The non-specific adsorption rate was calculated by comparing the peak area ratio of analyte to internal standard (IS) in the ultrafiltration sample and the negative control sample.

[0100] Serum samples were diluted with phosphate-buffered saline (PBS, 550 μL, pH 7.2–7.4) at a ratio of 1:1 (v / v). 1 mL of the mixture was transferred to a centrifuge filtration unit and centrifuged at 2000 rpm for 30 minutes at 37°C. More than 600 μL of the ultrafiltration solution was collected for subsequent sample extraction.

[0101] Sample extraction Activation and Equilibrium of Magnetic Beads First, the solvent in the commercial magnetic bead dispersion (50 mg / mL) was removed using a magnetic separator. 20 times the volume of methanol was added to the magnetic beads, and the beads were activated for 2 minutes. After removing the supernatant by magnetic separation, an equal volume of deionized water was added. d(H2O) , equilibrate for 2 min. Remove the supernatant by magnetic separation again, and finally resuspend the magnetic beads in 10 times the volume of deionized water to obtain a magnetic bead solution with a final concentration of 5 mg / mL.

[0102] Magnetic solid-phase extraction (MSPE) and in-situ derivatization This application systematically optimizes the magnetic solid phase extraction (MSPE) scheme to improve extraction efficiency and analytical performance. The adsorption effects of nine commercially available magnetic beads (MSi050 / Steroid MB, MSi050 / Steroid MB-Q, MSi050 / PLS, MPS10K / PLS, HLB, MPS10K / Steroid MB, MPS05K / Steroid MB, MSi050 / Steroid MB-T (MBT), and MGO) on androgens were compared.

[0103] Specifically, 600 μL of ultrafiltration solution containing the target androgen was mixed with different magnetic beads and incubated for a specific time to achieve efficient adsorption. After incubation, the supernatant was removed by magnetic separation, and QAO reagent was added for in-situ derivatization. The derivatization conditions were meticulously optimized to obtain the best reaction efficiency and mass spectrometry response. After derivatization, MSPE was performed, and the resulting supernatant was transferred to a sample vial for LC-MS / MS analysis.

[0104] Instruments and analytical conditions Chromatographic separation was performed using an LC-20AD high-performance liquid chromatography (HPLC) system connected in series with an AB Sciex 5500 QTrap mass spectrometer (Applied Biosystems, Foster City, California, USA), equipped with an electrospray ionization (ESI) source (TurboIonspray). The mass spectrometer was in positive ion mode, and derivatized androgens and internal standards (IS) were identified using multiple reaction monitoring (MRM) mode. The ion source temperature was set to 650 °C, the ion spray voltage to 5500 V, the nebulizer and heating gas pressures to 60 psi, the curtain gas pressure to 20 psi, and the collision-induced dissociation (CAD) gas to a medium intensity. Detailed optimized mass spectrometry (MS) parameters are available in [link to MS parameters]. Figure 2 .

[0105] Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) at a column temperature of 45 °C. Mobile phase A was 10 mM ammonium formate aqueous solution, and mobile phase B was acetonitrile containing 0.1% (v / v) formic acid. The flow rate was 0.32 mL / min, and the injection volume was 5 μL for quantitative analysis.

[0106] The gradient elution procedure is as follows: In the first 0-0.8 minutes, the volume percentage of mobile phase B was 10%. At 1.2 minutes, the volume percentage of mobile phase B increased to 20%; At 5.5 minutes, the volume percentage of mobile phase B increased to 22%. At the 7th minute, the volume percentage of mobile phase B increased to 25%; At 10.5 minutes, the volume percentage of mobile phase B increased to 38%. At the 11th minute, the volume fraction of mobile phase B increased to 95%. From 11.2 to 14 minutes, the volume percentage of mobile phase B was 100%. At 14.2-15 minutes, the volume percentage of mobile phase B was 10%.

[0107] Method Validation The LC-MS / MS method was validated according to the Clinical and Laboratory Standards Institute (CLSI-C62 A) guidelines, and key performance indicators of the method were comprehensively evaluated, including specificity, matrix effect, linearity, limit of quantitation (LLOQ), precision, accuracy, carryover, extraction recovery, and stability.

[0108] Assessment of endogenous interference and matrix effects To investigate endogenous interference, serum samples from six different subjects were collected. Endogenous steroid hormones were removed by activated carbon adsorption. Ultrafiltration solutions were obtained following the ultrafiltration steps described in the examples, and double-blank (DB), single-blank (SB), and lower limit of quantitation (LLOQ) samples were prepared. The specificity of the method was evaluated by observing the presence of interfering peaks at the retention times of the analyte and internal standard. Simultaneously, the matrix effect was assessed using the ultrafiltration solutions. Specifically, low and high concentrations of quality control working solutions were added to the ultrafiltration solution extract and deionized water, respectively. The coefficient of variation (CV%) of the ISMF for the six ultrafiltration solution samples was calculated using the "internal standard normalized matrix factor (ISMF)" as the primary evaluation parameter.

[0109] Calibration curve and linearity evaluation Calibration curves were plotted using eight non-zero calibrators at different concentrations. The concentration ranges, limits of quantitation (LLOQ), and quality control (QC) standard concentrations of nine analytes are summarized in [the table / document / reference]. Figure 1 A calibration curve was plotted with the peak area ratio of the analyte to the corresponding internal standard (IS) on the ordinate and the analyte concentration on the abscissa, using 1 / x. 2 Weights, calculate the correlation coefficient (r).

[0110] Precision, accuracy and carryover contamination assessment Precision and accuracy were assessed at five concentration levels: lower limit of quantitation (LLOQ) and four quality control (QC) levels. Freshly prepared LLOQ and QC samples were analyzed for three consecutive days, with six parallel samples per day. Intra-day and inter-day precision were expressed as coefficient of variation (CV%), and intra-day and inter-day accuracy were expressed as relative error (RE%).

[0111] To assess the carryover effect, double blank (DB) samples were injected immediately after the highest concentration calibration standard (ULOQ) was injected; the carryover effect was determined by comparing the peak area at the analyte retention time in the DB sample with the corresponding peak area in the LLOQ sample.

[0112] Extraction recovery rate assessment Extraction recoveries of the analyte were evaluated at four quality control (QC) levels, with three replicates per concentration level. The corresponding QC working solution was added to the samples before and after MSPE extraction, while the internal standard (IS) working solution was added after extraction to correct for variability introduced during derivatization and subsequent analysis. R1 was defined as the analyte to internal standard peak area ratio for the pre-extraction spiked sample, and R2 as the post-extraction spiked sample. Extraction recoveries were calculated as R2 / R1. Simultaneously, the recovery rate of the internal standard was evaluated at a single concentration level.

[0113] Stability assessment Considering the high affinity of androgens for serum proteins and the differences in binding rates of different analytes, serum quality control samples (SQCs) were prepared by adding high-concentration standard solutions to real serum samples. The concentration of these SQCs was 20 times the upper limit of quantitation (ULOQ) concentration in the ultrafiltration solution. Mixed serum samples were used to prepare SQCs to ensure consistency and facilitate statistical analysis.

[0114] The stability of free androgens in serum before ultrafiltration was assessed using SQC samples: SQC samples were placed at room temperature (RT) for 24 hours and at 4°C for 72 hours, respectively. After ultrafiltration, the concentration of free androgens was measured and compared with that of unstored SQC samples. The relative error (RE%) and coefficient of variation (CV%) were calculated.

[0115] In addition, the stability of QC1 and QC4 samples in ultrafiltration solution under different storage and treatment conditions was evaluated: the stability of low and high concentration QC samples in ultrafiltration solution after 72 hours at 4°C or 24 hours at room temperature (pretreatment stability), the stability of treated samples after 5 days at 4°C or 24 hours at room temperature (posttreatment stability), the stability of low and high concentration QC samples in ultrafiltration solution after 3 freeze-thaw cycles (-80°C to room temperature), and the long-term stability of low and high concentration QC samples in ultrafiltration solution after 2.5 months of storage at -80°C.

[0116] Statistical analysis Data acquisition and quantitative analysis were performed using Analyst 1.5.2 software. Excel was used for data processing and statistical analysis, and Origin Pro 2024 SR1 (version 10.1.0.178) was used to create charts of the raw data. Descriptive statistics are expressed as mean ± standard deviation (SD). The interquartile range (IQR) represents the middle 50% of the dataset and is calculated by subtracting the lower quartile (Q1) from the upper quartile (Q3). Differences between two groups were assessed using the Mann-Whitney U test. Statistical significance was defined as follows: NS, no significant difference; *p≤0.05; **p≤0.01; ***p≤0.001.

[0117] Results and Discussion Evaluation of non-specific adsorption and ultrafiltration efficiency To reduce the non-specific adsorption of analytes by the ultrafiltration membrane, various pretreatment methods were used to pretreat the regenerated cellulose membrane. The corresponding non-specific adsorption rates are shown in [Figure number missing]. Figure 3 The results showed that after rinsing with deionized water (Method 1), the internal standard (IS) peak shape was abnormal and an unknown peak appeared. Rinsing with 0.1 M sodium hydroxide (Method 2) led to a decrease in the non-adsorption rate of most analytes, indicating that this method was not optimal. Immersion in 1% BSA solution (Method 3) had poor applicability, with the recovery rate of 9 analytes being only 35%-65%, which may be due to the binding of androgens to BSA, consistent with the results after adding BSA to the negative control group.

[0118] Subsequent experiments evaluated the 5% KH2881 soaking (Method 4) and the two-step treatment of "KH2881 + glucocorticoids" (Method 5). Specifically, the recovery rate of 11OHA4 significantly increased to 144% after the addition of hydrocortisone. Similarly, the recovery rate of the negative control with added hydrocortisone increased by 160.7%, while the recovery rate of hydrocortisone treatment alone (without ultrafiltration) was 71.6%. This result is consistent with the literature report that "hydrocortisone can be non-enzymatically converted to 11OHA4," which interferes with the accurate quantification of 11OHA4. In contrast, the addition of other glucocorticoids did not significantly improve the analyte recovery rate and even led to a decrease in the recovery rate of testosterone (T) and dihydrotestosterone (DHT).

[0119] In summary, 5% KH2881 treatment (Method 4) can achieve analyte recoveries of 83.5%-112.8% with minimal impact on the quantification of free androgens, making it a feasible and practical pretreatment method.

[0120] After membrane pretreatment, serum samples were diluted with phosphate-buffered saline (PBS) at a ratio of 1:1 (v / v) to simulate human physiological conditions, and then centrifuged at 37°C at low speed to minimize interference from protein-bound androgens and ensure accurate quantification of free androgens.

[0121] Optimization of Magnetic Solid Phase Extraction (MSPE) and In-situ Derivatization Conditions The magnetic solid-phase extraction (MSPE) method developed in this application emphasizes environmental sustainability, requiring small sample volumes and low organic solvent usage. First, the extraction efficiency of nine commercially available magnetic beads on androgens from ultrafiltration solutions of the same concentration was compared, and the results are as follows: Figure 4 As shown. Although MPS05K / Steroid MB beads and HLB beads showed high signal response to the target analyte, the HLB beads had too large a signal standard deviation, and the MBT beads had too high a baseline signal, so they were both excluded. Due to their large specific surface area and excellent enrichment efficiency for steroidal compounds (including androgens), MSi050 / Steroid MB-T (MBT) beads were finally selected.

[0122] To optimize the amount of magnetic beads used, the effect of 0.02–2 mg MBT magnetic beads on the signal intensity of nine analytes was evaluated. The results are as follows: Figure 5 As shown, with the increase of magnetic bead dosage, the analyte signal intensity first gradually increased and then slightly decreased, while the signal-to-noise ratio showed a decreasing trend, possibly due to the extraction of more impurities. Therefore, the optimal balance point for magnetic bead dosage is 0.1-0.5 mg, and 0.5 mg was used in subsequent experiments.

[0123] In addition, the adsorption time was optimized, and the results are as follows: Figure 6 As shown, the signal intensity increases with adsorption time, reaching a peak at 15 minutes, and then shows no further increase. Therefore, 15 minutes was chosen as the adsorption time.

[0124] To optimize the elution conditions of MBT magnetic beads after androgen adsorption, the effects of elution with deionized water and 10%-30% methanol on sensitivity and matrix interference were compared. The results are as follows: Figure 7 As shown, the peak shapes of the nine analytes did not improve significantly after rinsing. Although the signal intensities of androstenedione (A4), 11β-hydroxyandrostenedione (11OHA4), and 11-ketoandrostenedione (11KA4) increased to varying degrees, the signal intensities of testosterone (T), dihydrotestosterone (DHT), 11β-hydroxytestosterone (11OHT), and 11-ketodihydrotestosterone (11KDHT) decreased compared to before rinsing. Considering the low concentration of free androgens in actual serum and to simplify the experimental procedure, the rinsing step was ultimately not used.

[0125] Furthermore, neutral androgens exhibit low ionization efficiency in electrospray ionization-tandem mass spectrometry (ESI-MS / MS), resulting in insufficient sensitivity for the detection of free androgens. However, oximeting of the androgen carbonyl group using QAO reagents can significantly improve sensitivity. Based on this, this application meticulously optimized the in-situ derivatization conditions, comparing the effects of four reaction matrices—50% methanol, 65% methanol, 80% methanol, and a mixed solvent of 10% methanol / 72% isopropanol (IPA)—on the derivatization efficiency. The results are as follows: Figure 8 As shown in the figure. The results indicate that, at the same reactant concentration, except for dehydroepiandrosterone (DHEA), the signal intensity of the analytes in 10% methanol and 72% isopropanol is lower than that in other solvents. The signal intensity of the analytes generally increases with increasing methanol concentration. Considering the sensitivity of all analytes, 80% methanol was selected as the reaction matrix for QAO derivatization.

[0126] The dosage of QAO reagent was optimized, and the results are as follows: Figure 9 As shown, with increasing QAO dosage, the signal intensity of most analytes increased and then plateaued, after which the signal intensity of all analytes decreased slightly. This may be related to the dilution effect caused by the increased reaction volume, precipitation caused by insufficient solubility of 200 mM QAO reagent, and the equilibrium of androgen mono- or di-substituted reactions. The final determined QAO reagent dosage was 2-8 μmol, and the reaction matrix was 80% methanol.

[0127] This application also evaluated the effect of derivatization temperature, and the results are as follows: Figure 10 As shown in the figure. Subsequent derivatization experiments were conducted at temperatures of 40-50℃.

[0128] Figure 11 The results showed that extending the reaction time could gradually improve the derivatization efficiency. Considering both efficiency and clinical applicability, 1.5 hours was selected as the derivatization time.

[0129] Figure 12 The effects of formic acid (FA) and acetic acid (AA), two Lewis acids, as catalysts were compared, and formic acid was found to have the highest catalytic efficiency. Further comparisons of different concentrations of formic acid and acetic acid, combined with column tolerance and oxime reaction efficiency, revealed that 8% formic acid was selected as the optimal catalyst.

[0130] Optimization of Chromatographic and Mass Spectrometry (MS / MS) Conditions When optimizing chromatographic and mass spectrometric conditions, QAO reagent was added to a series of single androgen standard solutions to obtain mass spectra of nine androgen-QAO derivatives. The carbonyl group in androgens reacts with the amino group (-ONH2) of the QAO reagent to form oxime derivatives, collectively referred to as androgen-QAO. Among the analyzed compounds, 11-ketotestosterone (11KT), 11-ketodihydrotestosterone (11KDHT), 11β-hydroxyandrostenedione (11OHA4), androstenedione (A4) contain dicarbonyl groups, while 11-ketoandrostenedione (11KA4) contains a tricarbonyl group. These androgens can not only form monosubstituted oxime derivatives but also undergo disubstituted or trisubstituted reactions with the QAO reagent.

[0131] Due to the complexity of carbonyl substitution reactions and the interference of isomers, most analytes preferentially choose monosubstituted derivatives as the parent ion. However, the disubstituted derivatives of 11β-hydroxyandrostenedione (11OHA4), androstenedione (A4), and 11-ketoandrostenedione (11KA4) are exceptions because they have better stability and sensitivity.

[0132] This application tested various C18 and fluorophenyl columns (including Poroshell 120 EC-C18, HSST3, and HSS PFP columns) to separate nine analytes and an internal standard (IS). Due to their excellent separation capabilities and sensitivity, the ACQUITY UPLC BEH C18 column was ultimately selected. Furthermore, the effectiveness of acetonitrile (ACN), methanol (MeOH), and a 1:1 (v / v) acetonitrile-methanol mixture as mobile phases was evaluated, revealing that acetonitrile offered superior separation efficiency and peak shape. Simultaneously, the buffer type, concentration, acidity, and gradient were optimized, ultimately determining a 15-minute gradient elution program for subsequent experiments.

[0133] Due to the presence of isomers and the fact that the charge ratio (m / z) difference of some analytes is less than 1 Da, cross-interference becomes a concern, which affects the selection of internal standard (IS) and the quantification of analyte peaks. Regarding the selection of internal standards, testosterone-d3 (T-d3), dehydroepiandrosterone-d6 (DHEA-d6), 11β-hydroxyandrostenedione-d4 (11OHA4-d4), and androstenedione-d7 (A4-d7) were retained, while 11-ketotestosterone-d3 (11KT-d3) was excluded. The reason is that after adding 11KT-d3, its monosubstituted or disubstituted derivatives would be subject to cross-interference from 11β-hydroxytestosterone-QAO (11OHT-QAO), 11-ketodihydrotestosterone-QAO (11KDHT-QAO), and even 11-ketodihydrotestosterone-double QAO (11KDHT-2QAO) and 11β-hydroxyandrostenedione-double QAO (11OHA4-2QAO). Furthermore, even without the addition of 11KT-d3, 11KT-d3-QAO, and 11KT-d3-2QAO, monosubstituted or disubstituted derivatives of 11OHA4-d4 were still affected by 11KDHT-2QAO. In contrast, 11OHA4-d4 experienced less interference than 11KT-d3 and could therefore be retained as an internal standard to partially correct for analytes retained more than 7 minutes earlier (such as 11OHA4-2QAO, 11KA4-2QAO, 11OHT-QAO, 11KDHT-QAO, and 11KT-QAO). This additional cross-interference further solidified the decision to exclude 11KT-d3 from the internal standard list.

[0134] Figure 13 Typical chromatograms of nine androgen-QAO derivatives are shown. Among them, testosterone-QAO (T-QAO) and dehydroepiandrosterone-QAO (DHEA-QAO) have mass-to-charge ratios of ( ). m / z The isomers of 403.3 both show peaks in each other's multiple reaction monitoring (MRM) ion channels; due to cross-interference, testosterone-d3-QAO ( m / z The concentration of testosterone-d3-QAO (m / z 406.4) is affected by dihydrotestosterone-QAO (DHT-QAO, m / z 405.4). To select suitable quantitative ion peaks, a single standard derivatization solution was injected: testosterone-d3-QAO showed two peaks at 9.64 min and 10.18 min, and dihydrotestosterone-QAO showed two peaks at 10.10 min and 10.54 min. Considering the interference between the testosterone-d3-QAO peak at 10.18 min and the dihydrotestosterone-QAO peak at 10.10 min, the peak at 9.64 min was selected as the quantitative peak for testosterone-d3-QAO, and the peak at 10.54 min was selected as the quantitative peak for dihydrotestosterone-QAO.

[0135] Furthermore, although dehydroepiandrosterone-QAO and testosterone-QAO differ in mass-to-charge ratio ( m / z (403.3) While both peaks appear in each other's ion channels, when dehydroepiandrosterone-QAO is injected alone, only a unique peak appears at 9.09 min, making it easily identifiable; testosterone-QAO shows two peaks at 9.64 min and 10.18 min, and dihydrotestosterone-QAO shows two peaks at 10.10 min and 10.54 min. Therefore, the peak at 9.09 min was selected as the quantitative peak for dehydroepiandrosterone-QAO, the peak at 9.64 min as the quantitative peak for testosterone-QAO, and the peak at 10.54 min as the quantitative peak for dihydrotestosterone-QAO in the mixed solution.

[0136] Furthermore, 11β-hydroxytestosterone (11OHT) and 11-ketodihydrotestosterone (11KDHT) are isomers, and their partial derivatization products may interfere with each other. To avoid interference between the 11OHT-QAO peak at 6.83 min and the 11KDHT-QAO peak at 7.13 min, the 11OHT-QAO peaks at 5.52 min and 6.02 min were selected as quantitative peaks. Although the isomers of 11-ketotestosterone (11KT) and 11β-hydroxyandrostenedione (11OHA4) can be distinguished after derivatization, the monosubstituted derivative of 11OHA4 at 7.22 min may still interfere with the monosubstituted product of 11KT at 7.10 min. Therefore, the peak at 6.09 min was ultimately selected as the quantitative peak of 11KT-QAO.

[0137] 11OHA4-2QAO ( m / z 266.1) and 11KA4-2QAO ( m / z The transition difference between the two androgen derivatives (265.3) was only 1 Da, and their retention times were similar (5.17 min and 5.29 min, respectively). Considering peak intensity, the 11OHA4-2QAO peak at 4.52 min and the 11KA4-2QAO peak at 4.85 min were selected as the quantitative peaks. The selection process for the quantitative peaks fully considered the effects of cross-interference, isomer interference, and similar mass-to-charge ratios, aiming to reduce interference and improve sensitivity, ensuring accurate quantification of each androgen derivative and meeting stringent analytical performance standards.

[0138] Method Validation Specificity was assessed using six ultrafiltration solutions of human serum pretreated with activated charcoal. Representative chromatograms of double-blank (DB), single-blank (SB), lower limit of quantitation (LLOQ) samples, and real patient samples are shown below. Figure 14 This application quantitatively analyzed the endogenous interference near the retention time of the analytes and internal standard (IS). The results showed that the interference of all analytes was less than 19.7%, which was negligible and did not affect the accurate determination of free androgen concentration.

[0139] The intra-day and inter-day precision / accuracy results of the nine analytes in three analytical batches are summarized in Figure 15 The intra-day and inter-day precision (expressed as CV%) ranged from -13.9% to 10.3%; the accuracy of calibration standards at four quality control (QC) levels ranged from 87.6% to 113.1%, and the accuracy of the lower limit of quantitation (LLOQ) remained stable between 103.7% and 108.2%. All measurement results were within an acceptable range of ≤15.0%, further confirming the reliability of the method; the linear fit correlation coefficient (r) exceeded 0.99, indicating excellent linearity.

[0140] Low and high concentrations of quality control working solutions were added to the ultrafiltration solution to assess matrix effects. This application carefully selected stable isotope-labeled internal standards (IS) to reduce matrix effects and ensure accurate quantification; however, some internal standards may interfere with other target analytes and were therefore excluded. The internal standard normalized matrix factor (ISMF) was calculated. Figure 16 The results showed that the coefficient of variation (CV%) of the matrix effect (ISMF) of the nine analytes ranged from 5.3% to 14.8%. According to the standard, a CV% of less than 15.0% indicates that the quantitative method for this sample is reliable.

[0141] Recovery rates of all target analytes were evaluated across the entire calibration range. The overall recovery rate of the nine androgens ranged from 95.1% to 105.2% (individual recoveries exceeding 100% were due to measurement error), with a variability of less than 6.5%. The recoveries of the four internal standards ranged from 82.4% to 99.0%. No significant bias trends were observed, further demonstrating the robustness and reliability of the established method and confirming its suitability for accurate quantification of androgens in ultrafiltration solutions.

[0142] The carryover effect was assessed in three analytical batches, and the carryover rate of all analytes was less than 20%, indicating negligible interference; this was also confirmed by precision and accuracy data.

[0143] Considering that freshly collected serum samples may not be ready for immediate ultrafiltration, this application evaluated the stability of serum samples before ultrafiltration. Figure 17 The results showed that ultrafiltration of serum samples stored at 4°C for 72 hours had minimal impact on the quantification of target free androgens; however, the relative error (RE%) of dehydroepiandrosterone (DHEA) after being stored at room temperature (RT) for 24 hours was -24.6%, which was consistent with the literature and may be due to the short elimination half-life (1-3 hours) of DHEA in serum.

[0144] In contrast, all analytes in the ultrafiltration solution remained stable after 72 hours of storage at 4°C or 24 hours at room temperature; all analytes in the extracted and derivatized samples also remained stable after 24 hours of storage at room temperature; and the nine androgens in the ultrafiltration solution remained stable after three freeze-thaw cycles and after long-term storage at -80°C for 2.5 months.

[0145] Clinical Application and Discussion Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women, with typical symptoms including infrequent menstruation (rare ovulation) and hyperandrogenemia (hyperandrogenemia). This application analyzed 86 female subjects, who were divided into two groups based on their anti-Müllerian hormone (AMH) levels: a “highly suspected PCOS group” with AMH levels greater than 10 ng / mL, and a “non-PCOS group” with AMH levels typically less than 1 ng / mL—AMH was chosen as the grouping criterion because it is a key biomarker for assessing ovarian reserve.

[0146] The distribution characteristics and statistical properties of nine free androgens in the serum of the two groups of subjects were comprehensively analyzed using the validated LC-MS / MS method. The results are as follows: Figure 18 The results showed significant differences between the two groups in the concentrations of free testosterone, 11β-hydroxyandrostenedione (11OHA4), and androstenedione (A4); however, in terms of 5α-dihydrotestosterone (DHT), the concentration of free DHT in 78 samples was below the lower limit of quantification (LLOQ), and 11-ketodihydrotestosterone (11KDHT) was not detected in 23 samples.

[0147] To determine whether the above results represent true physiological levels or are a methodological artifact, this application conducted further verification by adding a high-concentration quality control solution (SQC, with a concentration 20 times that of ULOQ working solution) to various serums. Figure 19 The results showed a significant increase in 11KDHT signal and a corresponding increase in free concentration. This result not only verified the reliability of the method but also confirmed that the level of free 11KDHT in clinical samples was indeed low.

[0148] Interestingly, the concentration of free dihydrotestosterone (DHT) remained low after the addition of SQC solution. To rule out analytical errors, the total DHT intensity in unfiltered male serum samples with added SQC solution was directly measured. The results showed that the total DHT intensity increased by approximately 3.14 times, consistent with expectations. As mentioned earlier, the ultrafiltration non-adsorption rate, magnetic bead extraction recovery rate, DHT stability data, and calibration curve linearity were all within acceptable ranges. These results further demonstrate that DHT detection is not affected by methodological factors.

[0149] The low concentration of free DHT may be related to its high affinity for sex hormone-binding globulin (SHBG) and other plasma proteins—previous studies have confirmed that the binding constant of DHT to these proteins is significantly higher than that of testosterone. This finding is consistent with previous research conclusions regarding the low free DHT / testosterone ratio in adult men, highlighting the physiological relevance of the results. In summary, these findings strongly suggest that the low concentration of free DHT is a genuine physiological phenomenon, rather than a methodological limitation, further supporting the hypothesis that DHT exists primarily in its protein-bound form in circulation.

[0150] The method described in this application exhibits excellent specificity and sensitivity, with a linear fitting correlation coefficient (r) greater than 0.99 and an extraction recovery rate higher than 95.1%. Precision and accuracy, measured by relative error and coefficient of variation, both meet the standards of the Clinical and Laboratory Standards Institute (CLSI). Stability tests confirm minimal analyte loss under typical clinical conditions. This validated method was applied to serum samples from 86 female patients, and the results showed significantly elevated levels of free testosterone, 11β-hydroxyandrostenedione, and androstenedione in patients with polycystic ovary syndrome (PCOS).

[0151] Compared with the method previously developed by the inventors (application number CN 2025101044132), the method described in this application can reach the lower limit of quantitation in pg / mL, and the sensitivity is significantly improved, with the sensitivity of the nine free androgens increasing by 10 to 125 times respectively.

[0152] This application successfully established a novel LC-MS / MS method for the simultaneous determination of nine free androgens in human serum. The method involves separating free androgens via ultrafiltration, followed by in-situ derivatization after magnetic solid-phase extraction (MSPE). The combination of MSPE and derivatization significantly improves detection sensitivity. This innovative method offers several advantages: reduced use of organic solvents (meeting environmental sustainability requirements), good reproducibility across different laboratories, and broad applicability. Furthermore, this application preliminarily reveals differences in the distribution of free androgens between PCOS patients and controls, which may provide important insights for identifying novel biomarkers for the diagnosis and management of PCOS.

Claims

1. A method for detecting multiple free androgens in serum, wherein, The method includes: The sample was subjected to ultrafiltration to obtain an ultrafiltration solution; Magnetic beads were added to the ultrafiltration solution, and androgens were extracted by magnetic solid-phase extraction. A derivatization reagent containing quaternary ammonium oxyamine was added to the extracted androgens to carry out an oxime reaction; The oxime-treated solution was injected into a high-performance liquid chromatography-tandem mass spectrometry system for detection.

2. The method according to claim 1, wherein, Before ultrafiltration, the ultrafiltration membrane is pretreated with an aminosilane coupling agent, preferably a monoaminosilane coupling agent, more preferably 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane or 4-aminobutyltriethoxysilane.

3. The method according to claim 2, wherein, The pretreatment includes soaking the ultrafiltration membrane in an aminosilane coupling agent solution for 1-5 hours, wherein the volume ratio of the coupling agent to the ultrafiltration solution is (1-10):

100.

4. The method according to claim 1, wherein, The magnetic beads are silicon-based steroid magnetic beads.

5. The method according to claim 1, wherein, Add 0.02-2 mg of magnetic beads, preferably 0.1-1 mg, to every 600 μL of the ultrafiltration solution.

6. The method according to claim 1, wherein, The adsorption time for magnetic solid phase extraction is 1-60 min, preferably 1-15 min.

7. The method according to claim 1, wherein, Add 1-20 μmol of quaternary ammonium oxygenase derivatizing reagent to each 600 μL ultrafiltration solution to carry out an oxime reaction, preferably 2-8 μmol of quaternary ammonium oxygenase derivatizing reagent.

8. The method according to claim 1, wherein, The reaction matrix for the oxime reaction is an aqueous methanol solution with a methanol concentration of 80% (V / V); the catalyst for the oxime reaction is an aqueous formic acid solution with a formic acid concentration of 8% (V / V).

9. The method according to claim 1, wherein, The reaction temperature of the oxime reaction is 20-60℃, preferably 40-50℃; the reaction time of the oxime reaction is 1.5-3h.

10. The method according to claim 1, wherein, The chromatographic column used in the high performance liquid chromatography was an ACQUITY UPLCBEH C18 column with dimensions of 2.1 × 50 mm and a diameter of 1.7 μm. or Gradient elution is performed in high-performance liquid chromatography 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, and mobile phase B is acetonitrile containing 0.1% (v / v) formic acid; or The gradient elution conditions were: elution temperature 45℃, flow rate 0.32 mL / min, and elution time 15 min; or 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.

Images