A method for detecting androgens using high performance liquid chromatography-tandem mass spectrometry

Abstract

The application discloses a method for detecting androgens by using high performance liquid chromatography-tandem mass spectrometry, wherein the method comprises the following steps: extracting androgens in a sample to be detected, and adding a derivatization reagent solution to perform in-situ derivatization treatment; and injecting the sample subjected to the in-situ derivatization treatment into a high performance liquid chromatography-tandem mass spectrometry system to perform detection; wherein the derivatization reagent solution contains a quaternary ammonium oxylamine derivatization reagent. The high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method provided by the application provides a sensitive, accurate and universal solution for analysis of various androgens, and overcomes the limitations of an electrochemiluminescence immunoassay (ECLIA) platform. The method of the application only needs a small amount of sample, and can meet the sensitivity requirement on a low-end device, and is a powerful tool for diagnosis and management of various endocrine diseases, and will be a better alternative to traditional immunoassay and high-end mass spectrometry methods.

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 classic androgens and 11-oxyandrogens. Background Technology

[0002] Androgen analysis is crucial for the diagnosis and management of various endocrine disorders, particularly polycystic ovary syndrome (PCOS), where hyperandrogenemia is a key characteristic. Androgen profiles are also essential for the diagnosis and differential diagnosis of many other endocrine disorders, such as adrenal diseases, congenital adrenal hyperplasia (CAH), and conditions associated with excess or deficiency of androgens.

[0003] The sources of androgens vary greatly. Classic androgens such as testosterone (T) and androstenedione (A4) are mainly produced by the ovaries, while 11-oxyandrogens (such as 11-ketotestosterone (11KT) and 11β-hydroxytestosterone (11β-OHT)) are primarily produced by the adrenal glands. Studies have shown that elevated levels of 11-oxyandrogens, including 11-ketotestosterone (11KT) and 11β-hydroxytestosterone (11β-OHT), are associated with metabolic disorders such as insulin resistance and dyslipidemia, which often co-occur with PCOS. Therefore, the ability to accurately measure various androgens from both ovarian and adrenal sources not only enhances the accuracy of diagnosing polycystic ovary syndrome (PCOS) but also holds great potential in the diagnosis, treatment, and differential diagnosis of various endocrine disorders.

[0004] Due to significant heterogeneity, androgen levels and metabolic dysfunction vary considerably among PCOS patients. Traditional diagnostic methods, such as electrochemiluminescence immunoassay (ECLIA), have limitations in specificity, sensitivity, and accuracy. To date, no ECLIA platform can measure the comprehensive androgen profile required for analysis, including 11-oxyandrogens such as 11-ketotestosterone (11KT), 11β-hydroxytestosterone (11OHT), and dihydrotestosterone (DHT). Cross-reactivity and the inability to distinguish similar steroid structures can lead to misleading results; therefore, accurate hormone levels are crucial for the diagnosis and management of complex endocrine disorders.

[0005] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) can simultaneously measure classical androgens and 11-oxyandrogens, providing a highly specific and sensitive method for androgen quantification. However, existing mass spectrometry methods require advanced, highly sensitive, and expensive equipment, which not all laboratories can afford. Existing low-end equipment often fails to achieve sufficient sensitivity, leading to unsatisfactory quantitative results. This significantly limits the widespread clinical application of LC-MS / MS methods.

[0006] Furthermore, many traditional methods require large sample volumes for testing, such as significant amounts of serum. However, when serum availability is limited, especially for pediatric or elderly patients, the test results are often unsatisfactory. Therefore, there is an urgent need for a testing method that can achieve high sensitivity and accuracy while requiring a small sample size, thereby improving its practicality and accessibility in various clinical settings.

[0007] In some prior studies, the inventors developed a method for detecting steroid hormones using high performance liquid chromatography-ion mobility differential mass spectrometry (patent publication number CN115541776), which is used to simultaneously determine multiple androgens in human serum, significantly improving mass spectrometry sensitivity and providing an excellent analytical tool for the diagnosis and management of polycystic ovary syndrome in clinical settings. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a high-performance liquid chromatography-tandem mass spectrometry method for detecting androgens with higher sensitivity than previously studied methods, and without the need to introduce ion mobility spectrometry. The method includes:

[0009] Androgens were extracted from the sample to be tested and in situ derivatized by adding a derivatization reagent solution.

[0010] The in-situ derivatized sample was injected into a high-performance liquid chromatography-tandem mass spectrometry system for detection.

[0011] The derivatizing reagent solution contains a quaternary ammonium oxide derivatizing reagent.

[0012] Furthermore, the concentration of the quaternary ammonium oxychloride derivatizing reagent is 10-100 mmol / L, preferably 30-80 mmol / L, and more preferably 40-60 mmol / L.

[0013] Furthermore, the derivatization reagent solution also contains methanol and / or Lewis acid.

[0014] Further, the concentration of the methanol is 60-95% (v / v), preferably 70-90% (v / v), more preferably 75-85% (v / v); and / or

[0015] The Lewis acid is selected from formic acid and / or acetic acid, preferably formic acid; or

[0016] The concentration of the Lewis acid is 0.5-5% (v / v), preferably 1-3% (v / v).

[0017] Furthermore, the in-situ derivatization temperature is 20-70℃, preferably 30-60℃, and more preferably 40-50℃; or

[0018] The in-situ derivatization time is greater than 1 hour, preferably greater than 1.5 hours.

[0019] Furthermore, androgens were extracted from the test sample using magnetic solid-phase extraction.

[0020] Furthermore, MGO is preferably used as a magnetic adsorbent, and the concentration of MGO is 0.1-2.5 mg / mL, preferably 0.2-2.0 mg / mL, and more preferably 1.0-2.0 mg / mL.

[0021] Further, in the high-performance liquid chromatography, gradient elution is performed using a mixed mobile phase consisting of mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of ammonium formate, mobile phase B is acetonitrile, and mobile phases A and B further contain formic acid at a concentration of 0.1% (v / v); or

[0022] The chromatographic column used in the high-performance liquid chromatography was a Poroshell EC-C18 column with dimensions of 150 mm × 3.0 mm and a diameter of 2.7 μm.

[0023] Furthermore, the gradient elution conditions are as follows: elution temperature is 40℃, flow rate is 0.4 mL / min; during the gradient elution process, the volume ratio of mobile phase B in the mixed mobile phase gradually increases from the initial ratio and then returns to the initial ratio.

[0024] Furthermore, the steroid hormone is selected from T, DHEA, A4, DHT, 11KT, 11OHT, 11OHA4, 11KA4, and 11KDHT.

[0025] The high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method described in this application provides a sensitive, accurate, and universal solution for the analysis of various androgens, overcoming the limitations of the electrochemiluminescence immunoassay (ECLIA) platform. This method requires only a very small sample size and can meet sensitivity requirements even on lower-end equipment, making it a powerful tool for the diagnosis and management of various endocrine diseases and a superior alternative to traditional immunoassays and high-end mass spectrometry methods. Attached Figure Description

[0026] Figure 1 The effect of the amount of magnetic adsorbent on the mass spectrometry response of different test androgens;

[0027] Figure 2 The effects of different derivatizing reagents on the mass spectrometry response of the tested androgens under different conditions are as follows:

[0028] 2(A) The derivatization temperature was 30℃, and the derivatization time was 6h.

[0029] 2(B) The derivatization temperature was 40℃, and the derivatization time was 6h.

[0030] 2(C) The derivatization temperature was 50℃, and the derivatization time was 6 hours.

[0031] 2(D) The derivatization temperature was 60℃ and the derivatization time was 6h;

[0032] Figure 3 It is the mass spectrometric fragmentation pathway of androgen-QAO derivatives;

[0033] Figure 4 The optimization of conditions for deriving serum androgens includes:

[0034] Figure 4 (A) is the derived temperature optimization.

[0035] Figure 4 (B) is a derivative time optimization.

[0036] Figure 4 (C) represents a comparison of the catalytic effects of different Lewis acids;

[0037] Figure 5 These are chromatograms of different samples after derivatization with quaternary ammonium oxychloride derivatization reagent, where: (A) blank matrix, (B) blank matrix with internal standard added, (C) sample with lower limit of quantitation, and (D) actual serum sample from the patient.

[0038] Figure 6 This is a comparison of serum androgen test results between two different groups of patients, as shown in the figure:

[0039] * indicates a statistically significant difference (P<0.05).

[0040] ** indicates a statistically significant difference (P<0.01).

[0041] *** indicates a highly statistically significant difference (P<0.001).

[0042] **** represents an extremely significant statistical difference (P<0.0001);

[0043] Figure 7 The calibration curve ranges of the target analyte and internal standard, the limit of quantitation / quality control sample concentration, and the key mass spectrometry / mass spectrometry conditions in the examples are:

[0044] Figure 8 The results of intraday and intraday precision and accuracy tests;

[0045] Figure 9 This is the result of serum matrix effect and recovery rate assessment;

[0046] Figure 10 This is the result of the stability assessment of the analyte;

[0047] Figure 11 This is a schematic diagram of the in-situ derivatization process for magnetic solid phase extraction of serum samples. Detailed Implementation

[0048] 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.

[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] 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.

[0051] This application develops a highly sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method for the simultaneous determination of nine androgens in human serum, including four classical androgens and five 11-oxyandrogens, providing a comprehensive androgen profile. This method is helpful in the diagnosis and identification of a range of diseases.

[0052] In this application, androgens can be selected from T, DHEA, A4, DHT, 11KT, 11OHT, 11OHA4, 11KA4, and 11KDHT. This androgen testing combination not only has significant practical value in diagnosing polycystic ovary syndrome, but can also be extended to other endocrine disorders, such as congenital adrenal hyperplasia, adrenal tumors, and androgen deficiency, and has potential applications in some non-endocrine diseases related to metabolic health.

[0053] The method for determining androgens described in this application includes:

[0054] Androgens were extracted from serum samples and derivatized by adding a derivatization reagent solution containing quaternary ammonium oxychloride reagent; the derivatized sample was then injected into a high-performance liquid chromatography-tandem mass spectrometry system for detection.

[0055] This application employs magnetic solid-phase extraction (MSPE) to extract androgens from serum samples, and then uses in-situ derivatization to process the extracted androgens. Specifically, a serum sample is taken, an internal standard solution and a saturated ammonium chloride solution are added, a magnetic adsorbent is added, the sample is allowed to stand for a period of time, the sample is placed on a magnetic rack, the supernatant is poured off, and the magnetic adsorbent is retained.

[0056] After magnetic solid-phase extraction (MSPE), the derivatization reagent was directly added to the magnetic adsorbent for reaction. Specifically, the derivatization reagent was added to the residual adsorbent and vortexed to form a suspension. Considering sensitivity and sample preparation speed, the suspension was then incubated for a period of time before being placed on a magnetic rack for further MSPE, and the clear solution containing the derivatization product was analyzed.

[0057] In some implementations, magnetic graphene oxide (Fe3O4@GO, MGO) is selected as the magnetic adsorbent at a concentration of 0.1-2.5 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2.0 mg / mL, 2.2 mg / mL, and 2.4 mg / mL.

[0058] In some preferred embodiments, the concentration of MGO is 0.2-2.0 mmol / L.

[0059] In some preferred embodiments, the concentration of MGO is 1.0-2.0 mmol / L.

[0060] In some preferred embodiments, the concentration of MGO is 1.6 mmol / L.

[0061] The quaternary ammonium oxyamine (QAO) derivatizing reagent used in this application possesses a permanently charged mass spectrometry tag and an aminooxy group that reacts with steroid carbonyl groups, thereby significantly improving ionization efficiency (complete ionization). This allows the method described in this application to detect low-abundance androgens, including those undetectable by electrochemiluminescence immunoassay (ECLIA). After comparing various derivatizing reagents, the inventors found that the QAO derivatizing reagent exhibits excellent sensitization effects for most androgens, especially for very low concentrations of androgens such as T, DHT, 11OHT, 11KT, and 11KDHT. In other words, the QAO-containing derivatizing reagent used in this application can significantly improve 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. The method described in this application requires only 100 μL of serum sample to detect the above-mentioned androgen combination. Compared with many existing technologies, the sample volume requirement is significantly reduced. Therefore, the method described in this application has great advantages when dealing with limited sample volumes.

[0062] This application uses quaternary ammonium oxygenase derivatizing reagents for in-situ derivatization, which essentially utilizes oxime reaction to derivatize androgens to obtain oxime-treated androgen products. 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 and QAO·Cl halides, where the Br or Cl ions do not affect the oxime-treatment of androgens.

[0063] In some embodiments of this application, QAO halides are used as derivatizing agents.

[0064] In some embodiments, the concentration of the quaternary ammonium oxychloride derivatizing agent is 10-100 mmol / L, for example, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, 80 mmol / L, 85 mmol / L, 90 mmol / L, 95 mmol / L, or 100 mmol / L.

[0065] In some preferred embodiments, the concentration of the quaternary ammonium oxyamine derivatizing agent is 30-80 mmol / L.

[0066] In some preferred embodiments, the concentration of the quaternary ammonium oxyamine derivatizing agent is 40-60 mmol / L.

[0067] In some preferred embodiments, the concentration of the quaternary ammonium oxyamine derivatizing agent is 50 mmol / L.

[0068] In some embodiments, the derivatizing reagent solution further comprises methanol and / or a Lewis acid. The Lewis acid may be selected from formic acid or acetic acid.

[0069] In some embodiments, the matrix is ​​selected as a methanol solution with a concentration of 60-95% (v / v), such as 60% (v / v), 65% (v / v), 70% (v / v), 75% (v / v), 80% (v / v), 85% (v / v), 90% (v / v), and 95% (v / v).

[0070] In some preferred embodiments, the concentration of the methanol solution is 70-90% (v / v).

[0071] In some preferred embodiments, the concentration of the methanol solution is 75-85% (v / v).

[0072] In some preferred embodiments, the concentration of the methanol solution is 80% (v / v).

[0073] In some embodiments, the derivatizing reagent solution further comprises formic acid, the concentration of which can be selected from 0.5-5% (v / v), for example, 0.5% (v / v), 1% (v / v), 1.5% (v / v), 2% (v / v), 2.5% (v / v), 3% (v / v), 3.5% (v / v), 4% (v / v), 4.5% (v / v), 5% (v / v).

[0074] In some preferred embodiments, the concentration of formic acid is 1-3% (v / v).

[0075] In some preferred embodiments, the concentration of formic acid is 2% (v / v).

[0076] The process of incubating the suspension is the process of in-situ derivatization of androgens. The derivatization temperature can be selected from 20-70℃, for example, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃.

[0077] In some preferred embodiments, the derivatization temperature is 30-60°C.

[0078] In some preferred embodiments, the derivatization temperature is 40-50°C.

[0079] The time for in situ derivatization of androgens is greater than 1 hour, such as 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, or even longer.

[0080] In some preferred schemes, the derivatization time is greater than 1.5 hours.

[0081] High performance liquid chromatography-mass spectrometry (HPLC-MS / MS) is a detection technique that combines chromatography and mass spectrometry, that is, separating samples by high performance liquid chromatography before mass spectrometry.

[0082] In some embodiments, the mass spectrometry is a two-stage mass spectrometer. Those skilled in the art will recognize that optional HPLC systems and tandem mass spectrometers can be used in the scheme of this application. For example, in subsequent embodiments of this application, a CalQuant-S LC-MS / MS liquid chromatography-mass spectrometry system (Hangzhou, China) from KELA Spectrometry was used for analysis.

[0083] In some implementations, the HPLC uses a liquid chromatography column with a C18 stationary phase (C18 liquid chromatography column).

[0084] In some embodiments, the chromatographic column is an octadecylsilane-bonded porous silica column.

[0085] 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. For example, the column can be an ACQUITY UPLC BEH C18 column (50×2.1mm, 1.7μm), a Poroshell EC-C18 column (50×2.1mm, 2.7μm), or a Poroshell EC-C18 column (150×2.1mm, 2.7μm).

[0086] In some implementations, the preferred chromatographic column is a Poroshell EC-C18 column (150 × 2.1 mm, 2.7 μm).

[0087] Those skilled in the art will understand that any mobile phase that enables adequate separation and good peak shape of the steroid hormones to be measured can be used in the scheme of this application. In some embodiments, a Poroshell EC-C18 column (150×2.1mm, 2.7μm) is used for chromatographic separation, and 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 proportion, and then returning to the initial proportion.

[0088] In some embodiments, the mobile phase A comprises an aqueous solution of ammonium formate, and the mobile phase B comprises acetonitrile.

[0089] In some embodiments, the concentration of ammonium formate in the mobile phase A is 5-15 mmol / L, such as 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, etc.

[0090] In some preferred embodiments, the concentration of the ammonium formate is 10 mmol / L.

[0091] In some embodiments, mobile phase A and mobile phase B further comprise an acid for adjusting the pH value.

[0092] In some implementations, the acid is a weak acid.

[0093] In some embodiments, the acid is formic acid or acetic acid.

[0094] In some embodiments, the acid is formic acid, and the content is 0.1% (v / v).

[0095] 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.

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

[0097] Stage 1: Mobile phase B is maintained at a low proportion for a period of time, allowing the analyte in the sample to accumulate at the column head and be separated from highly polar impurities in the sample. For example, mobile phase B is maintained at a proportion of 1-20% for 0-5 minutes, such as proportions of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%, and maintained for 0 minutes, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, and 5 minutes.

[0098] Stage 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. For example, the proportion of mobile phase B is 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.

[0099] 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, such as 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 min.

[0100] 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, such as 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.

[0101] The proportion of mobile phase B (organic phase) will be adjusted in each stage, which may be related to different chromatographic columns and systems, and requires detailed optimization.

[0102] Stage 1: The volume percentage of mobile phase B is 5%, and it is maintained for 1 minute;

[0103] Phase 2: Within 16 minutes, the volume percentage of mobile phase B is gradually increased to 90% in 5 stages.

[0104] Stage 3: The volume of mobile phase B is 90%, and it is maintained for 1.5 min;

[0105] Stage 4: The volume percentage of mobile phase B is reduced to 5% and maintained for 1.5 min.

[0106] 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.

[0107] In some implementations, chromatographic separation is performed using a Poroshell EC-C18 column (150×2.1mm, 2.7μm) at 40°C.

[0108] In some implementations, the flow rate of the mobile phase is 0.4 mL / min.

[0109] In some implementations, the autosampler temperature is maintained at 8°C.

[0110] In some implementations, the injection volume is 25-30 μL.

[0111] 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.

[0112] 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.

[0113] Example

[0114] Material sources used in the embodiments

[0115] 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), 11-ketotestosterone (11KT), and 11-ketotestosterone-d3 (11KT-d3) 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).

[0116] HPLC-grade acetonitrile, methanol, and formic acid were purchased from Thermo Fisher Scientific (Fair Lawn, NJ, USA). Ammonium formate was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Hydroxylamine hydrochloride (HAHC), N,N,N-triethyl-2-hydrazyl-2-oxoethyl ammonium chloride (TEAH), N,N-dibutyl-N-(2-hydrazyl-2-oxoethyl)-butyl-1-ammonium chloride (DBAH), 2-(2-hydrazyl-2-oxoethyl)-isoquinoline-2-hydrazine bromide (IQHB), Girard reagent P (GP), and Girard reagent T (GT) were purchased from Bidepharm. Co. Ltd (Shanghai, PR China), (O-(3-trimethylammoniumpropyl)hydroxylamine) bromide (QAO·Br) and 2-(aminooxy)ethyl-N,N,N-trimethylammonium (ATM) trifluoromethanesulfonate were purchased from Pharmaron Inc. (Beijing, China).

[0117] Clinical sample sources used in the examples

[0118] The clinical samples used in this application were all obtained from the Reproductive Medicine Center of Peking University Third Hospital. The examples included 107 female patients of reproductive age, aged 20 to 49 years (mean age 34.85 ± 7.23 years), all of whom presented with clinical symptoms of infertility. Blood samples were collected using separating gel coagulation tubes on days 2 to 5 of the menstrual cycle. Biochemical indicators, testosterone (T), androstenedione (A4), anti-Müllerian hormone (AMH), fasting blood glucose, and fasting insulin levels were measured using chemiluminescent immunoassay. The remaining serum was stored at -80°C before subsequent analysis.

[0119] This study has been approved by the Medical Ethics Committee of Peking University Third Hospital (Approval No.: IRB00006761-M2022236), and all data were obtained using Analyst 1.5.2 software. The analyses in this study were performed using Excel and GraphPadPrism 8.0 software.

[0120] Preparation of steroid-free matrix

[0121] In this application, since all target androgens are endogenous substances, human serum cannot be used as a blank matrix for preparing calibrators and quality control (QC) samples. To eliminate these endogenous androgens, serum was treated with activated carbon adsorption as an alternative blank matrix for subsequent analysis.

[0122] Specifically, endogenous steroids were removed using serum / activated charcoal at a ratio of 20 / 1 (v / w). The mixture of serum and activated charcoal was vortexed for 3 min, stored at 4°C for 48 hours, then centrifuged at 4574 g for 10 min and filtered through a 0.2 μm membrane to obtain steroid-free serum, which was used as a blank matrix for subsequent analysis.

[0123] Preparation of calibration curves and quality control samples

[0124] Individual stock solutions of the analytes are prepared in methanol to obtain stock solutions of appropriate concentrations (some of which are pre-prepared solutions). Mixed secondary stock solutions are prepared by diluting the stock solutions with methanol. Calibrators and quality control (QC) standards are prepared by continuous dilution of the mixed secondary stock solutions with methanol. These stock solutions are then diluted in methanol to prepare mixed working solutions. Internal standard (IS) stock solutions are prepared using d3-T, d4-11OHA4, d3-11KT, and d6-DHEA.

[0125] All samples and standards are stored at -80°C before use.

[0126] The calibration range for each analyte is set with reference to actual clinical use.

[0127] Preparation of calibration curve samples and quality control samples: Add 10 μL of working solution to 190 μL of blank matrix to prepare 8 non-zero calibrators, LLOQ (lower limit of quantitation) samples and 4 QC standards.

[0128] Example 1: Extraction and in-situ derivatization of androgens from serum

[0129] Androgens were extracted from serum samples using magnetic solid-phase extraction (MSPE). 100 μL of serum sample, calibrator, or quality control standard was added, along with 5 μL of internal standard solution and 50 μL of saturated ammonium chloride solution. Then, 500 μL of Fe3O4@GO (MGO, 2 mg / mL) was added as a magnetic adsorbent. After standing for 20-30 min, the sample was placed on a magnetic rack, and the supernatant was carefully decanted. Next, 100 μL of derivatization reagent solution (containing 5 mM quaternary ammonium oxychloride (QAO) in 80% (v / v) methanol solution containing 2% (v / v) formic acid) was added to the remaining adsorbent, and the mixture was vortexed for 1 min. Considering sensitivity and sample preparation speed, the suspension was then incubated at 40-50 °C for 1.5-2 h before being placed on a magnetic rack for further MSPE. The clear solution containing the derivatized product was transferred to a vial for analysis.

[0130] Example 2: High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS / MS) Analysis

[0131] Samples were analyzed using a CalQuant-S liquid chromatography-mass spectrometry (LC-MS) system. All target analytes were detected in positive ionization mode, with a source temperature of 600°C, nebulizer and heating gas both set to 60 psi, and ion spray voltage of 5500 V. Quantitative analysis was performed using multiple reaction monitoring (MRM) mode; details of MRM transitions can be found in [link to MRM documentation]. Figure 7 .

[0132] Data acquisition and analysis were performed using mass spectrometry / mass spectrometry (Kailai Spectrometry).

[0133] Chromatographic separation was performed using a Poroshell EC-C18 column (150 × 2.1 mm, 2.7 μm, Agilent) at 40 °C. The mobile phase consisted of 10 mM ammonium formate aqueous buffer (mobile phase A) and acetonitrile (mobile phase B), both containing 0.1% (v / v) formic acid. The flow rate was 0.4 mL / min, the autosampler temperature was maintained at 8 °C, and the injection volume was 25–30 μL.

[0134] The specific gradient elution process is as follows:

[0135] During the first minute, the volume percentage of mobile phase B was maintained at 5%.

[0136] Subsequently, within 2 minutes, the volume percentage of mobile phase B increased to 21%;

[0137] Subsequently, within 4 minutes, the volume percentage of mobile phase B was further increased to 28%, and then maintained for 2 minutes;

[0138] Subsequently, within 3 minutes, the volume percentage of mobile phase B continued to increase by 1% to 29%, and was then maintained for 1 minute.

[0139] Subsequently, within 1.5 min, the volume percentage of mobile phase B was increased to 45%, and then maintained for 0.5 min;

[0140] Subsequently, within 2 minutes, the volume percentage of mobile phase B increased sharply from 45% to 90%, and was then maintained for 1.5 minutes to clean the column.

[0141] Subsequently, the volume percentage of mobile phase B decreased to 5% within 0.1 min, and the system equilibrated for 1.4 min.

[0142] Example 3: Optimization of the Magnetic Solid Phase Extraction (MSPE) Process

[0143] Based on the inventors' prior research, this study utilized magnetic graphene oxide (Fe3O4@GO, MGO) to purify and enrich nine androgens in serum. The dosage of MGO was re-optimized, and the effect of the amount of magnetic adsorbent on the mass spectrometry response of different androgens is shown in the following results. Figure 1 As shown.

[0144] according to Figure 1 It can be seen that for the various androgens described in this application, the mass spectrometry response peak is highest when the dosage of MGO is 1.6 mg. Therefore, 1.6 mg of MGO was used in subsequent experiments.

[0145] Example 4: Optimization of in-situ derivatization conditions

[0146] Optimization of derivatization conditions included optimization of the type of derivatizing reagent, reaction matrix, derivatization temperature and time, catalyst type, and concentration of the derivatizing reagent. The results are as follows: Figure 2-4 As shown.

[0147] First, the types of derivatization reagents were screened, and the sensitizing effects of different quaternary ammonium hydrazine, quaternary ammonium oxychloride, and hydroxylamine hydrochloride derivatization reagents were compared, including eight derivatization reagents: TEAH, DBAH, QAO, ATM, HAHC, GT, GP, and IQHB. As shown in Figure 2, when the derivatization temperature is between 30 and 60℃ (with a derivatization reaction time of 6 hours to ensure sufficient reaction), QAO derivatization reagent showed the best sensitizing effect for most androgens. Especially for very low concentrations of T, DHT, 11OHT, 11KT, and 11KDHT, the sensitizing effect of QAO was more significant than that of other derivatization reagents. Figure 2The results showed that, compared with the GP derivatization reagent used in previous studies (derivatization conditions: 50℃, 6h, complete reaction), the sensitivity of other androgens (except A4) using the QAO derivatization reagent was increased by 1.7 to 147.7 times. Considering that the concentration of A4 in serum is relatively high compared with other androgens, its sensitivity is not a limiting factor for mass spectrometry detection. In other words, for androgen profiling, using QAO as the derivatization reagent is more advantageous.

[0148] from Figure 2 It can be seen that, given sufficient derivatization time, regardless of temperature, quaternary ammonium oxide (QAO) is the most effective derivatization reagent for improving mass spectrometry response for most analytes. Figure 3 Data shows that for most analytes derivatized using QAO, the fragmentation mode is very simple, with most androgens producing a single, strong, and specific daughter ion, thereby improving sensitivity and selectivity and reducing the possibility of cross-interference.

[0149] like Figure 4 As shown, this application also optimizes the in-situ derivatization temperature and time. Considering both reaction efficiency and time, the in-situ derivatization of androgens can be carried out at a temperature of 40-50℃ for a reaction time exceeding 1.5 hours.

[0150] This application also compared the responses in different reaction matrices and found that 80% (v / v) methanol produced the highest response. Figure 4 As shown in (C), 2% (v / v) formic acid performed slightly better among the various Lewis acids used as catalysts. Therefore, 80% (v / v) methanol was used as the reaction matrix and 2% (v / v) formic acid was used as the catalyst in subsequent experiments.

[0151] Example 5: Optimization of HPLC-MS / MS conditions

[0152] As a mass spectrometry tagging reagent, all analytes are permanently positively charged after derivatization with quaternary ammonium oxychloride, which greatly improves mass spectrometry sensitivity. This application optimizes the multiple reaction monitoring (MRM) conditions, with results as follows: Figure 7 As shown.

[0153] The derivatization process described in this application can distinguish some androgen isomers, such as 11OHA4 and 11KT (mass-to-charge ratios of 266.2→236.7 and 417.3→358.2, respectively). However, for T and DHEA, the resulting fragment ions are identical, therefore separation is required before MS / MS analysis. Some androgens, after derivatization with quaternary ammonium oxides, form more than one isomer. These isomers have the same fragment ions and produce specific and strong mass spectrometric signals, but they produce different chromatographic peaks on the column, and these peaks are difficult to separate at the baseline. To achieve sufficient separation, the inventors tested several chromatographic columns, including the ACQUITY UPLC BEH C18 column (50×2.1mm, 1.7μm), the Poroshell EC-C18 column (50×2.1mm, 2.7μm), and the Poroshell EC-C18 column (150×2.1mm, 2.7μm). By comparing the separation results, this application selected the Poroshell EC-C18 column (150×2.1mm, 2.7μm), and its representative chromatogram is shown below. Figure 5 As shown.

[0154] Example 6 Method Verification

[0155] The selectivity of the above method was assessed using six different batches of steroid-free serum. The results showed that the endogenous interference of all analytes was less than 20%, and the interference of all internal standards was less than 5%, indicating that the endogenous interference of the above protocol was negligible.

[0156] The calibration range was set according to clinical needs, with the LLOQ range for different target androgens between 0.005-1 ng / mL. The limit of quantitation (S / N>10) or limit of detection (S / N>3) of the method described in this application is significantly lower than the LLOQ set according to clinical needs, indicating that the method described in this application has high sensitivity and can expand the calibration range. The calibration curve was established using eight non-zero calibrators with a weighting factor of 1 / x. 2 The proposed method employs linear fitting for all analytes, with correlation coefficients greater than 0.9900. In all acceptable analytical batches, the accuracy of the calibrators ranges from 85% to 115%.

[0157] At LLOQ and four QC levels, measurements were repeated six times on three different dates, each day, to assess intra-day and inter-day precision and accuracy. Results are as follows: Figure 8As shown. The intra- and inter-assay precision of LLOQ (expressed as coefficient of variation, C% (V / V)) is ≤9.7%, and the relative error percentage (RE%) for all analytes is between -8.2% and 7.5%. For QC samples, the intra- and inter-assay precision for all analytes is ≤13.8%, and the relative deviation is between -7.4% and 11.3%.

[0158] The matrix effect was assessed using six different batches of steroid-free serum. Results are shown in [Figure number missing]. Figure 9 In these six batches of serum, the C% (V / V) of the internal standard normalized matrix effect (ISMF) was less than 8.0% at low QC levels and less than 12.2% at high QC levels, both meeting the detection requirements. The recoveries of the analyte and internal standard were evaluated at four QC levels. The results showed that the overall recoveries of all target androgens ranged from 41.8% to 80.2% across the entire quantification range, with C% (V / V) less than 9.9% between different concentrations.

[0159] Residue is a key indicator for assessing the robustness of a assay. To assess residue, a blank sample was injected immediately after the upper limit of quantitation (ULOQ) was injected, and the peak area of ​​the blank sample was compared with the peak area of ​​the LLOQ. The results showed that the residue of all target androgens was less than 12.6%, meaning that the residue during the analysis was negligible.

[0160] Stability was evaluated at low and high QC levels, and the results are as follows: Figure 10 As shown, all analytes are stable in serum for at least 4 hours at room temperature and 32 hours at 8°C after extraction. All androgens can withstand three freeze / thaw cycles and remain stable for at least 34 days at -80°C.

[0161] Example 7 Method Application

[0162] The inventors collected fasting serum from 107 patients who routinely underwent oral glucose tolerance tests at the applicant's reproductive center. These individuals were typically obese or at high risk for metabolic diseases and were suspected of having polycystic ovary syndrome (PCOS). Patients were divided into two groups based on serum anti-Müllerian hormone (AMH) levels: AMH > 10 and AMH < 0.5. The nine androgens in these samples were analyzed using the detection method described in this application to determine whether the differences in androgen levels were significant. The test results are as follows: Figure 6 As shown.

[0163] Figure 6The results show that there are highly significant differences between the two groups for T, A4, DHT, DHEA, and 11KA4 (P<0.0001); there is a statistically significant difference between the two groups for 11OHA4 (P<0.05); while there is no difference for 11KT; the detection results for 11OHT and 11KDHT are lower than those for LLOQ.

[0164] The high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method proposed in this application, combined with in-situ derivatization technology, optimizes derivatization reagents and multiple detection conditions, enabling the simultaneous quantitative analysis of nine androgens in human serum, including classical androgens and 11-oxyandrogens. This method can be used even with lower-end mass spectrometers because it has high sensitivity and specificity.

[0165] Compared to traditional immunoassay techniques (such as electrochemiluminescence immunoassay, ECLIA) that cannot accurately measure all types of androgens, the method described in this application has significant advantages. It can be used not only for the diagnosis and classification of polycystic ovary syndrome (PCOS), but also has great application value in the diagnosis and differentiation of other endocrine disorders. For example, congenital adrenal hyperplasia (CAH) is characterized by adrenal hyperplasia and excessive secretion of adrenal-derived androgens. Accurate measurement of androgens such as 11KT and 11β-OHT is crucial for differentiating CAH from PCOS, as both diseases may present with androgen excess, but they have distinct underlying causes and treatment approaches. Furthermore, accurate detection of 11KDHT, a potent adrenal-derived androgen, may provide further clues to understanding excessive adrenal androgen secretion and its role in other diseases such as Cushing's syndrome or adrenal tumors.

[0166] The technical features of the above embodiments can be combined arbitrarily. 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.

[0167] 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 detecting androgens using high performance liquid chromatography-tandem mass spectrometry, wherein, The method includes: Androgens were extracted from the test sample using magnetic solid phase extraction and then derivatized in situ using a derivatization reagent solution. The in-situ derivatized sample was injected into a high-performance liquid chromatography-tandem mass spectrometry system for detection. The derivatizing reagent solution contains a quaternary ammonium oxyamine derivatizing reagent; 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 an aqueous solution of ammonium formate and mobile phase B is acetonitrile, and mobile phases A and B also contain formic acid at a concentration of 0.1% (v / v). The chromatographic column used in the high-performance liquid chromatography was a Poroshell EC-C18 column with dimensions of 150 mm × 3.0 mm and 2.7 μm. The gradient elution conditions are as follows: elution temperature is 40 °C, and flow rate is 0.4 mL / min; during the gradient elution process, the volume ratio of mobile phase B in the mixed mobile phase gradually increases from the initial ratio and then returns to the initial ratio. The specific gradient elution process is as follows: During the first minute, the volume percentage of mobile phase B remained at 5%. Subsequently, within 2 minutes, the volume percentage of mobile phase B increased to 21%; Subsequently, within 4 minutes, the volume percentage of mobile phase B was further increased to 28%, and then maintained for 2 minutes. Subsequently, within 3 minutes, the volume percentage of mobile phase B continued to increase by 1% to 29%, and was maintained for 1 minute. Subsequently, the volume fraction of mobile phase B was increased to 45% within 1.5 min, and then maintained for 0.5 min. Subsequently, within 2 minutes, the volume percentage of mobile phase B increased sharply from 45% to 90%, and was maintained for 1.5 minutes to clean the column. Subsequently, within 0.1 min, the volume percentage of mobile phase B decreased to 5%, and it equilibrated for 1.4 min. The androgens mentioned are selected from testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, 11-ketotestosterone, 11β-hydroxytestosterone, 11β-hydroxyandrostenedione, 11-ketoandrostenedione, and 11-ketodihydroandrostone.

2. The method according to claim 1, wherein, The concentration of the quaternary ammonium oxide derivatizing reagent is 10-100 mmol / L.

3. The method according to claim 1, wherein, The concentration of the quaternary ammonium oxide derivatizing reagent is 30-80 mmol / L.

4. The method according to claim 1, wherein, The concentration of the quaternary ammonium oxide derivatizing reagent is 40-60 mmol / L.

5. The method according to claim 1, wherein, The concentration of the quaternary ammonium oxide derivatizing reagent is 80-100 mmol / L.

6. The method according to claim 1, wherein, The derivatization reagent solution also contains methanol and / or Lewis acid.

7. The method according to claim 6, wherein, The concentration of methanol is 60-95% (v / v); and / or The Lewis acid is selected from formic acid and / or acetic acid; or The concentration of the Lewis acid is 0.5-5% (v / v).

8. The method according to claim 6, wherein, The concentration of methanol is 70-90% (v / v).

9. The method according to claim 6, wherein, The concentration of methanol is 75-85% (v / v).

10. The method according to claim 6, wherein, The Lewis acid is formic acid.

11. The method of claim 6, wherein, The concentration of the Lewis acid is 1-3% (v / v).

12. The method according to claim 1, wherein, The temperature for in-situ derivatization is 20-70 °C; or The in-situ derivatization time was greater than 1 hour.

13. The method according to claim 1, wherein, The temperature for in-situ derivatization is 30-60 °C.

14. The method according to claim 1, wherein, The temperature for in-situ derivatization is 40-50 °C.

15. The method according to claim 1, wherein, The in-situ derivatization time was greater than 1.5 h.

16. The method according to claim 1, wherein, MGO is used as a magnetic adsorbent, and the concentration of MGO is 0.1-2.5 mg / mL.

17. The method of claim 16, wherein, The concentration of MGO is 0.2-2.0 mg / mL.

18. The method of claim 16, wherein, The concentration of MGO is 1.0-2.0 mg / mL.

Images