Method for detecting androgen by high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention relates to a method for detecting androgen by high performance liquid chromatography-tandem mass spectrometry, which comprises the following steps: extracting androgen in a sample to be detected, and adding a derivatization reagent solution for in-situ derivatization treatment; injecting the sample subjected to in-situ derivatization treatment into a high performance liquid chromatography-tandem mass spectrometry system for detection; wherein the derivatization reagent solution contains a quaternary ammonium oxygen amine derivatization reagent. According to the present invention, the high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method provides the sensitive, accurate and universal solution for the analysis of a variety of androgens, and overcomes the limitation of the ECLIA platform. According to the method, only a very small amount of samples are needed, the sensitivity requirement can also be met on lower-end equipment, and the method is a powerful tool for diagnosis and management of various endocrine diseases and will serve as a better alternative scheme for traditional immunoassay and high-end mass spectrometry methods.

Description

Technical Field

[0001] The present application relates to a detection method combining liquid chromatography and tandem mass spectrometry, and in particular to a method for simultaneously detecting multiple classical androgens and 11-oxygenated androgens. Background Art

[0002] Androgen analysis is essential for the diagnosis and management of various endocrine disorders, especially polycystic ovary syndrome (PCOS), where hyperandrogenemia is a key feature. Androgen profiles are also essential in the diagnosis and differentiation of many other endocrine disorders, such as adrenal disease, congenital adrenal hyperplasia (CAH), and conditions associated with androgen excess or deficiency.

[0003] The sources of androgens vary greatly. For example, classic androgens such as testosterone (T) and androstenedione (A4) are mainly produced by the ovaries, while the main source of 11-oxygenated androgens (such as 11-ketotestosterone (11KT) and 11β-hydroxytestosterone (11β-OHT)) is the adrenal gland. Studies have shown that the increase of 11-oxygenated androgens such as 11-ketotestosterone (11KT) and 11β-hydroxytestosterone (11β-OHT) is associated with metabolic dysfunctions including insulin resistance and dyslipidemia, which often occur simultaneously with PCOS. Therefore, the ability to accurately measure various androgens, including those from the ovaries and adrenal glands, can not only enhance the ability to accurately diagnose PCOS, but also has great potential in the diagnosis, treatment and differentiation of various endocrine diseases.

[0004] Due to the strong heterogeneity, the androgen and metabolic dysfunction in different PCOS patients vary greatly. Traditional diagnostic methods, such as electrochemiluminescence immunoassay (ECLIA), have great limitations in specificity, sensitivity and accuracy. So far, no electrochemiluminescence immunoassay platform can measure the androgen spectrum required for comprehensive analysis of 11-oxygenated androgens including 11-ketotestosterone (11KT), 11β-hydroxytestosterone (11OHT), and dihydrotestosterone (DHT). Cross-reactions and the inability to distinguish between similar steroid structures can also lead to misleading results. Therefore, accurate hormone levels are crucial for the diagnosis and management of complex endocrine diseases.

[0005] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) can simultaneously measure classical androgens and 11-oxygenated androgens, providing a highly specific and sensitive method for androgen quantification. However, existing mass spectrometry methods require advanced, highly sensitive and expensive equipment, but not all laboratories can be equipped with the most advanced instruments, and existing low-end equipment is difficult to achieve sufficient sensitivity, resulting in unsatisfactory quantitative results, which greatly limits the widespread clinical application of LC-MS / MS methods.

[0006] In addition, many traditional methods require a large amount of samples for testing, such as a large amount of serum, but when serum is limited, especially for pediatric or elderly patients, the test results are often not ideal. Therefore, there is an urgent need for a detection 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 previous 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 the sensitivity of mass spectrometry and providing an excellent analytical tool for the diagnosis and management of polycystic ovary syndrome in a clinical setting. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a method for detecting androgens with high performance liquid chromatography-tandem mass spectrometry, which has higher sensitivity than the previously studied method, and does not require the introduction of ion mobility spectrometry, wherein the method comprises:

[0009] Extracting androgen from the sample to be tested, and adding a derivatization reagent solution to perform in-situ derivatization treatment;

[0010] The sample treated with in situ derivatization was injected into a high performance liquid chromatography-tandem mass spectrometry system for detection;

[0011] Wherein, the derivatization reagent solution contains a quaternary ammonium oxide amine derivatization reagent.

[0012] Furthermore, the concentration of the quaternary ammonium oxide amine derivatization agent is 10-100 mmol / L, preferably 30-80 mmol / L, and more preferably 40-60 mmol / L.

[0013] Furthermore, the derivatization reagent solution further comprises 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] Further, the temperature of the in-situ derivatization is 20-70°C, preferably 30-60°C, preferably 40-50°C; or

[0018] The time for in situ derivatization is greater than 1 h, preferably greater than 1.5 h.

[0019] Furthermore, magnetic solid phase extraction is used to extract androgens from the test samples.

[0020] Further, MGO is preferably used as the magnetic adsorbent, and the concentration of the 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, a mixed mobile phase consisting of a mobile phase A and a mobile phase B is used for gradient elution, the mobile phase A is an aqueous solution of ammonium formate, the mobile phase B is acetonitrile, the mobile phase A and the mobile phase B also include formic acid, and the formic acid concentration is 0.1% (v / v); or

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

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

[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 multiple androgens, overcoming the limitations of the electrochemiluminescence immunoassay (ECLIA) platform. The method of this application requires only a very small amount of sample and can meet the sensitivity requirements on lower-end equipment. It is a powerful tool for the diagnosis and management of multiple endocrine diseases and will serve as a better alternative to traditional immunoassays and high-end mass spectrometry methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the effect of the amount of magnetic adsorbent on the mass spectrometric response of different androgens to be measured;

[0027] Figure 2 It is the effect of different derivatization reagents on the mass spectrometric response of the androgen to be tested under different conditions, among which:

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

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

[0030] 2(C) The derivatization temperature was 50°C and the derivatization time was 6 h.

[0031] 2(D) derivatization temperature was 60 °C and derivatization time was 6 h;

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

[0033] Figure 4 The conditions for deriving serum androgens are optimized, where:

[0034] Figure 4 (A) is the derivative temperature optimization,

[0035] Figure 4 (B) is the derivative time optimization,

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

[0037] Figure 5 The chromatograms of different samples after derivatization with quaternary ammonium oxide amine derivatization reagents are shown in Figure 1, including: (A) blank matrix, (B) blank matrix with internal standard added, (C) quantitative limit sample, and (D) actual serum sample of the patient;

[0038] Figure 6 This is a comparison of the serum androgen test results of two groups of different patients. In the figure:

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

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

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

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

[0043] Figure 7 is the calibration curve range of the target analyte and internal standard, the lower limit of quantitation / quality control sample concentration, and the key mass spectrometry / mass spectrometry conditions in the embodiments;

[0044] Figure 8 It is the intra-day and inter-day precision and accuracy test results;

[0045] Fig. 9 It is the result of serum matrix effect and recovery evaluation;

[0046] Fig.10 It is the result of stability assessment of the substance to be tested;

[0047] Fig.11 This is a schematic diagram of the in situ derivatization process of magnetic solid phase extraction of serum samples. DETAILED DESCRIPTION

[0048] The specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments or examples set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present application and to enable the scope of the present application to be fully communicated to those skilled in the art.

[0049] In the present application, “% (v / v)” represents the volume percentage of the substance. For example, “the concentration of 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 solution.

[0050] The quaternary ammonium oxide amine derivatizing agent refers to an aminooxy group that can react with the carbonyl group of the steroid. In this application, "quaternary ammonium oxide amine" and "QAO" can be used interchangeably.

[0051] The present application develops a highly sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method for simultaneously determining nine androgens in human serum, including four classical androgens and five 11-oxygenated androgens, which can provide a comprehensive androgen spectrum. The method is helpful for diagnosing and identifying a series of diseases.

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

[0053] The method for determining androgen described in the present application includes:

[0054] Androgens are extracted from serum samples, and a derivatization reagent solution containing a quaternary ammonium oxide reagent is added for derivatization treatment; the derivatized samples are injected into a high performance liquid chromatography-tandem mass spectrometry system for detection.

[0055] The present application uses magnetic solid phase extraction (MSPE) to extract androgens from serum samples, and uses an in situ derivatization method to treat 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, and the sample is allowed to stand for a period of time, and the sample is placed on a magnetic rack, and the supernatant is poured out, leaving the magnetic adsorbent.

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

[0057] In some embodiments, magnetic graphene oxide (Fe3O4@GO, MGO) is selected as the magnetic adsorbent and its concentration is 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 oxide amine derivatization reagent used in the present application has a permanently charged mass spectrometry label and an aminooxy group that can react with the carbonyl group of the steroid, thereby greatly improving the ionization efficiency (complete ionization), so that the method described in the present application can detect low-abundance androgens, including those that cannot be detected by electrochemiluminescence immunoassay (ECLIA). After comparing a variety of derivatization reagents, the inventors found that for most androgens, the sensitization effect of the QAO derivatization reagent is very good, especially for androgens such as T, DHT, 11OHT, 11KT, 11KDHT, etc. with very low concentrations, the sensitization effect of the QAO derivatization reagent is more significant. In other words, the derivatization reagent containing QAO used in the present application can significantly improve the sensitivity of the mass spectrometer. This method enables the detection of a variety of androgens even in laboratories equipped with lower-end mass spectrometry systems, providing powerful help for the diagnosis and management of diseases. The method described in the present application only requires 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 the present application has great advantages when dealing with limited sample volumes.

[0062] The present application uses a quaternary ammonium oxide amine derivatization reagent for in-situ derivatization, which is actually to use an oximation reaction to derivatize androgen to obtain an oximation product of androgen. Those skilled in the art should understand that all quaternary ammonium oxide amine derivatization reagents containing aminooxy groups can achieve the present application, regardless of the acid radical ion bonded to the aminooxy group, such as QAO·Br, QAO·Cl in QAO halides, etc., in which Br or Cl ions do not affect the oximation of androgen.

[0063] In some embodiments of the present application, QAO halide is used as a derivatization agent.

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

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

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

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

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

[0069] In some embodiments, the matrix is ​​selected as a methanol solution, and its concentration can be selected from 60-95% (v / v), for example 60% (v / v), 65% (v / v), 70% (v / v), 75% (v / v), 80% (v / v), 85% (v / v), 90% (v / v), 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 derivatization reagent solution further comprises formic acid, and its concentration 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, and the derivatization temperature can be selected to be 20-70°C, for example, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C.

[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, for example, 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 embodiments, the derivatization time is greater than 1.5 h.

[0081] High performance liquid chromatography-mass spectrometry (HPLC-MS / MS) is a detection technology that combines chromatography with mass spectrometry, that is, the sample is separated by high performance liquid chromatography before mass spectrometry.

[0082] In some embodiments, the mass spectrum is a secondary mass spectrum. Those skilled in the art will appreciate that optional HPLC systems and tandem mass spectrometry can be used in the present application. For example, in the subsequent examples of the present application, CalQuant-S LC-MS / MS liquid-mass spectrometry system (Hangzhou, China) of Kelley Spectrum was used for analysis.

[0083] In some embodiments, the chromatographic column used in HPLC is a liquid chromatography column having a carbon 18 stationary phase (C18 liquid chromatography column).

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

[0085] Those skilled in the art should know that any brand of octadecylsilane bonded porous silica gel column can be used for the scheme of the present application. Only because of the differences between different brands of chromatographic columns, the elution process will be different when performing high performance liquid chromatography separation. The chromatographic column includes, but is not limited to, commercially available core-shell octadecylsilane bonded columns from manufacturers such as Agilent, Waters, Thermo, etc. For example, the chromatographic column can select ACQUITY UPLC BEH C18 column (specifications are 50×2.1mm, 1.7μm), Poroshell EC-C18 column (specifications are 50×2.1mm, 2.7μm) or Poroshell EC-C18 column (specifications are 150×2.1mm, 2.7μm).

[0086] In some embodiments, the preferred chromatography column is a Poroshell EC-C18 column (with dimensions of 150×2.1 mm, 2.7 μm).

[0087] Those skilled in the art should know that any mobile phase that can achieve sufficient separation and good peak shape of the steroid hormone to be tested can be used in the scheme of the present application. In some embodiments, a Poroshell EC-C18 column (specifications are 150×2.1 mm, 2.7 μm) is used for chromatographic separation, and the mixed mobile phase for gradient elution is composed of mobile phase A and mobile phase B. The gradient elution process using the mixed mobile phase is that the volume proportion of the mobile phase B in the mixed mobile phase gradually increases from the initial proportion and then returns to the initial proportion.

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

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

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

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

[0092] In some embodiments, 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] During the gradient elution process, the volume proportion of mobile phase B in the mixed mobile phase can be gradually increased to elute the analyte, then increased to a high proportion to clean the chromatographic column, and finally restored to the initial state equilibrium, and the above process can be repeatedly cycled during the analysis process. Those skilled in the art should know that during the gradient elution process, the change in the ratio of A to B in the mobile phase is a continuous process, and according to the specific implementation of the gradient elution, those skilled in the art can stay at any ratio for a certain period of time according to the operating conditions at the time, or adjust the speed of change between any two ratios.

[0096] In some embodiments, the gradient elution process is (the ratios described in the elution process are all volume ratios):

[0097] Stage 1: Mobile phase B is maintained at a lower ratio for a period of time so that the analyte in the sample is enriched at the head of the chromatographic column and can be separated from the impurities with high polarity in the sample. For example, mobile phase B is maintained at a ratio of 1-20% for 0-5 minutes, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 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, 5 minutes.

[0098] Stage 2: According to the properties of the analyte in the sample, gradually increase the proportion of mobile phase B in multiple stages within a certain period of time. For example, within 10-40 minutes, increase the proportion of mobile phase B to 20-100% in 3-10 stages, so that the analytes of different polarities in the sample are eluted one by one, and obtain a better separation effect for accurate quantification. For example, increase the proportion of mobile phase B to 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% in 3, 4, 5, 6, 7, 8, 9, 10 stages.

[0099] Stage 3: After the analytes are basically eluted, the mobile phase B is maintained at a ratio of 85-100% for a certain period of time to fully flush the impurities in the chromatographic column and ensure that some weakly polar substances in the sample can also be eluted, so as not to affect the next injection. For example, the mobile phase B is maintained at a ratio of 85%, 90%, 95% or 100% for 1-10 minutes, such as 1min, 1.5min, 2min, 2.5min, 3min, 3.5min, 4min, 4.5min, 5min, 5.5min, 6min, 6.5min, 7min, 7.5min, 8min, 8.5min, 9min, 9.5min, 10min.

[0100] Stage 4: The proportion of mobile phase B is restored to the initial proportion, that is, the proportion of mobile phase B in stage 1, and maintained for a certain period of time for equilibrium to ensure the reproducibility of each injection. For example, mobile phase B is maintained at the proportion of stage 1 for 1-10 minutes, such as 1min, 1.5min, 2min, 2.5min, 3min, 3.5min, 4min, 4.5min, 5min, 5.5min, 6min, 6.5min, 7min, 7.5min, 8min, 8.5min, 9min, 9.5min, 10min.

[0101] There will be some adjustments in the proportion of mobile phase B (organic phase) in each stage. This may be related to different chromatographic columns and systems and requires detailed optimization.

[0102] Stage 1: Mobile phase B accounts for 5% by volume, maintained for 1 min;

[0103] Stage 2: Within 16 min, the volume proportion of mobile phase B was gradually increased to 90% in 5 stages;

[0104] Stage 3: Mobile phase B accounts for 90% of the volume, maintained for 1.5 min;

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

[0106] In some embodiments, the time in each stage and the volume ratio of mobile phase B in the above embodiments can be adjusted according to actual conditions.

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

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

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

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

[0111] The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, the technical solution of the present application can be subjected to a variety of simple modifications, including combining various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed in the present application and belong to the protection scope of the present application.

[0112] The exemplary embodiments of the present application are described below in conjunction with the accompanying drawings, which include various details of the embodiments of the present application to facilitate understanding, and the results reflect the technical effects that can generally be achieved by the implementation methods covered by the present application.

[0113] Example

[0114] Sources of materials used in the examples

[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-ketodihydroandrostenone (11KDHT) were purchased from Steraloids (Newport, RI, USA), and 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 for HPLC 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-hydrazino-2-oxoethylammonium chloride (TEAH), N,N-dibutyl-N-(2-hydrazino-2-oxoethyl)-butyl-1-ammonium chloride (DBAH), 2-(2-hydrazino-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] Sources of clinical samples used in the examples

[0118] The clinical samples used in this application were all taken from the Reproductive Medicine Center of Peking University Third Hospital. The embodiment included 107 female patients of childbearing age, aged between 20 and 49 years old (average age was 34.85 ± 7.23 years old), all of whom had clinical symptoms of infertility. On the 2nd to 5th day of the menstrual cycle, blood samples were collected using separation gel coagulation tubes, and biochemical indicators, testosterone (T), androstenedione (A4), anti-Mullerian hormone (AMH), fasting blood glucose and fasting insulin levels were determined by chemiluminescent immunoassay, and 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 number: IRB00006761-M2022236), and all data were obtained using Analyst 1.5.2 software. The analysis in this study was performed using Excel software and GraphPadPrism8.0 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. In order to eliminate these endogenous androgens, activated carbon adsorption was used to treat serum as a substitute blank matrix for subsequent analysis.

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

[0123] Preparation of calibration curve and quality control samples

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

[0125] All samples and standards were stored at -80°C until use.

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

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

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

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

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

[0131] The samples were analyzed using a CalQuant-S LC / MS system. All target analytes were detected in positive ionization mode, with the source temperature set to 600°C, the nebulizer and heater gas set to 60 psi, and the ion spray voltage set to 5500 V. Multiple reaction monitoring (MRM) mode was used for quantitative analysis. For details on the MRM transitions, see Figure 7 .

[0132] Data acquisition and analysis were performed by MS / MS (Kelley Spectroscopy).

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

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

[0135] In the first 1 min, the volume proportion of mobile phase B was maintained at 5%;

[0136] Then, within 2 min, the volume proportion of mobile phase B increased to 21%;

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

[0138] Then, within 3 min, the volume proportion of mobile phase B continued to increase by 1% to 29%, and then maintained for 1 min;

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

[0140] Then, within 2 min, the volume proportion of mobile phase B increased sharply from 45% to 90% and then maintained for 1.5 min to clean the column;

[0141] Then, within 0.1 min, the volume proportion of mobile phase B was reduced to 5% and equilibrated for 1.4 min.

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

[0143] Based on the inventors' previous research, this study used magnetic graphene oxide (Fe3O4@GO, MGO) to purify and enrich nine androgens in serum, and re-optimized the dosage of MGO. The results of the effect of the amount of magnetic adsorbent on the mass spectrometry response of different androgens to be tested are shown in Figure 2. Figure 1 shown.

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

[0145] Example 4 Optimization of in situ derivatization conditions

[0146] The optimization of derivatization conditions includes the optimization of the type of derivatization reagent, reaction substrate, derivatization temperature and time, catalyst type and derivatization reagent concentration. Figure 2-4 shown.

[0147] First, the types of derivatization reagents were screened, and the sensitization effects of different quaternary ammonium hydrazine, quaternary ammonium oxide and hydroxylamine hydrochloride derivatization reagents were compared, including 8 derivatization reagents including TEAH, DBAH, QAO, ATM, HAHC, GT, GP, and IQHB. As can be seen from Figure 2, when the derivatization temperature is between 30 and 60°C (the derivatization reaction time is 6 hours to ensure sufficient reaction), for most androgens, the sensitization effect of using QAO derivatization reagent is the best. Especially for very low concentrations of T, DHT, 11OHT, 11KT, 11KDHT, etc., the sensitization effect of QAO is more significant than that of other derivatization reagents. Figure 2The results showed that compared with the GP derivatization reagent used in previous studies (derivation conditions: 50°C, 6h, full reaction), the sensitivity of other androgens except A4 was improved by QAO derivatization reagent by 1.7 to 147.7 times. Considering that the concentration of A4 in serum is higher than that of other androgens, its sensitivity is not a limiting factor for mass spectrometry detection. In other words, it is more advantageous to use QAO as a derivatization reagent for androgen spectrum detection.

[0148] from Figure 2 It can be seen that given sufficient derivatization reaction time, regardless of the temperature, quaternary ammonium oxide (QAO) is the most effective derivatization reagent for improving mass spectrometric response for most analytes. Figure 3 The data showed that for most analytes derivatized with QAO, the fragmentation patterns were very simple, with most androgens producing single, strong and specific product ions, thus improving sensitivity and selectivity and reducing the potential for cross-interference.

[0149] like Figure 4 As shown, the present application also optimizes the in situ derivatization temperature and time. Taking the reaction efficiency and time into consideration, the in situ derivatization of androgens is carried out at a temperature of 40-50° C. and the reaction time is more than 1.5 h.

[0150] The present application also compares the responses in different reaction matrices, and the results show that 80% (v / v) methanol produces the highest response. Figure 4 C shows that among the various Lewis acids used as catalysts, 2% (v / v) formic acid has a slightly better effect. Therefore, in subsequent experiments, 80% (v / v) methanol was used as the reaction substrate and 2% (v / v) formic acid was used as the catalyst.

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

[0152] As a mass spectrometry labeling reagent, all analytes are permanently positively charged after derivatization with quaternary ammonium oxide amine, which greatly improves the mass spectrometry sensitivity. This application optimizes the multiple reaction monitoring conditions, and the results are as follows Figure 7 shown.

[0153] The derivatization treatment described in the present application can distinguish the isomers of some androgens, such as 11OHA4 and 11KT (mass-to-charge ratios of 266.2→236.7 and 417.3→358.2, respectively), but for T and DHEA, the fragment ions produced are the same, so they need to be separated before MS / MS analysis. Some androgens will form more than one isomer after derivatization with quaternary ammonium oxide amines, and their fragment ions are the same and can produce specific and strong mass spectrometry signals, but they will produce different chromatographic peaks on the chromatographic column, and these peaks are difficult to achieve baseline separation. In order to achieve adequate separation, the inventors tested several chromatographic columns, including ACQUITY UPLC BEH C18 column (50×2.1mm, 1.7μm), Poroshell EC-C18 column (50×2.1mm, 2.7μm) and Poroshell EC-C18 column (150×2.1mm, 2.7μm). By comparing the separation results, the present application selected the Poroshell EC-C18 column (150×2.1mm, 2.7μm). Its representative chromatogram is shown in FIG. Figure 5 shown.

[0154] Example 6 Method Verification

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

[0156] The calibration range is set according to clinical needs, and the LLOQ range of different target androgens is between 0.005-1ng / mL. The lower limit of quantification (S / N>10) or detection limit (S / N>3) of the method described in this application is significantly lower than the LLOQ set according to clinical needs, which shows that the method described in this application has high sensitivity and can expand the calibration range. The establishment of the calibration curve uses 8 non-zero calibrants with a weighting factor of 1 / x 2 The scheme of the present application used linear fit for all analytes, and the correlation coefficients were all greater than 0.9900. In all acceptable analytical batches, the accuracy of the calibrants was between 85% and 115%.

[0157] At LLOQ and 4 QC levels, the assay was repeated 6 times on three different days to assess intra-day and inter-day precision and accuracy. Figure 8As shown. The intra- and inter-assay precision (expressed as coefficient of variation, C% (V / V)) of LLOQ was ≤9.7%, and the relative error percentage (expressed as RE%) of all analytes was between -8.2% and 7.5%. For QC samples, the intra- and inter-assay precision of all analytes was ≤13.8%, and the relative deviation was between -7.4% and 11.3%.

[0158] Matrix effects were evaluated using six different batches of steroid-free serum and the results are shown in Fig. 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 the low QC level and less than 12.2% at the high QC level, both of which met the detection requirements. The recoveries of the analytes and internal standards were evaluated at four QC levels, and the results showed that the total recoveries of all target androgens in the entire quantitative range were between 41.8% and 80.2%, and the C% (V / V) between different concentrations was less than 9.9%.

[0159] Carryover is a key indicator for evaluating the robustness of the assay. To evaluate carryover, a blank sample was injected immediately after the upper limit of quantitation (ULOQ) injection, and the peak area of ​​the blank sample was compared with the peak area of ​​the LLOQ. The results showed that the carryover of all target androgens was less than 12.6%, which means that the carryover during the analysis can be ignored.

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

[0161] Example 7 Method Application

[0162] The inventor collected fasting serum from 107 patients who were routinely undergoing oral glucose tolerance tests at the reproductive center of the applicant's unit. These patients were usually obese patients or high-risk groups for metabolic diseases and were suspected of being related to polycystic ovary syndrome (PCOS). According to the serum anti-Mullerian hormone (AMH) value, the patients were divided into two groups: 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 difference in androgen levels was significant. The test results were as follows: Figure 6 shown.

[0163] Figure 6From the results, it can be seen that there were extremely significant differences between the two groups for T, A4, DHT, DHEA and 11KA4 (P<0.0001); there was a statistical difference between the two groups for 11OHA4 (P<0.05); but there was no difference for 11KT; the detection results of 11OHT and 11KDHT were lower than LLOQ.

[0164] The high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method proposed in this application is combined with in situ derivatization technology. By optimizing the derivatization reagent and optimizing multiple detection conditions at the same time, it can be used to simultaneously perform quantitative analysis of nine androgens in human serum, including classical androgens and 11-oxygenated androgens. This method can be used even when using a lower-end mass spectrometer because it has high sensitivity and specificity.

[0165] Compared with traditional immunoassay techniques (such as electrochemiluminescence immunoassay, ECLIA) that cannot accurately measure all types of androgens, the method described in the present application has significant advantages. It can not only be used for the diagnosis and classification of polycystic ovary syndrome, but also has great application value in the diagnosis and differentiation of other endocrine diseases. 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 to distinguishing congenital adrenal hyperplasia from polycystic ovary syndrome, because both diseases may show excessive androgens, but they have completely different underlying causes and treatment approaches. In addition, accurate detection of 11KDHT, a potent androgen derived from the adrenal glands, may provide more clues for a deeper understanding of adrenal androgen excess and its role in other diseases such as Cushing's syndrome or adrenal tumors.

[0166] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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 above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A method for detecting androgen by high performance liquid chromatography-tandem mass spectrometry, wherein: The method comprises: Extracting androgen from the sample to be tested, and adding a derivatization reagent solution to perform in-situ derivatization treatment; The sample treated with in situ derivatization was injected into a high performance liquid chromatography-tandem mass spectrometry system for detection; Wherein, the derivatization reagent solution contains a quaternary ammonium oxide amine derivatization reagent.

2. The method according to claim 1, wherein: The concentration of the quaternary ammonium oxide amine derivatization agent is 10-100 mmol / L, preferably 30-80 mmol / L, and more preferably 40-60 mmol / L.

3. The method according to claim 1, wherein: The derivatization reagent solution further comprises methanol and / or a Lewis acid.

4. The method according to claim 3, wherein: The concentration of the methanol is 60-95% (v / v), preferably 70-90% (v / v), more preferably 75-85% (v / v); and / or The Lewis acid is selected from formic acid and / or acetic acid, preferably formic acid; or The concentration of the Lewis acid is 0.5-5% (v / v), preferably 1-3% (v / v).

5. The method according to claim 1, wherein: The temperature of the in situ derivatization is 20-70°C, preferably 30-60°C, more preferably 40-50°C; or The time for in situ derivatization is greater than 1 h, preferably greater than 1.5 h.

6. The method according to claim 1, wherein: Magnetic solid phase extraction was used to extract androgens from the test samples.

7. The method according to claim 6, wherein: Preferably, MGO is used as the 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.

8. The method according to claim 1, wherein: Using a mixed mobile phase consisting of a mobile phase A and a mobile phase B for gradient elution in a high performance liquid chromatography, wherein the mobile phase A is an aqueous solution of ammonium formate, the mobile phase B is acetonitrile, the mobile phase A and the mobile phase B further comprise formic acid, and the formic acid concentration is 0.1% (v / v); or The chromatographic column used in the high performance liquid chromatography is a Poroshell EC-C18 chromatographic column with a specification of 150 mm×3.0 mm and 2.7 μm.

9. The method according to claim 8, wherein: The gradient elution conditions are as follows: the elution temperature is 40° C. and the flow rate is 0.4 mL / min. During the gradient elution process, the volume proportion of the mobile phase B in the mixed mobile phase gradually increases from the initial proportion and then returns to the initial proportion.

10. The method of claim 1, wherein the steroid hormone is selected from the group consisting of T, DHEA, A4, DHT, 11KT, 11OHT, 11OHA4, 11KA4, and 11KDHT.

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