CN106568852B - Steroid hormone marker related to idiopathic male sterility in serum and detection method and application thereof - Google Patents
Steroid hormone marker related to idiopathic male sterility in serum and detection method and application thereof Download PDFInfo
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Abstract
The invention belongs to the fields of analytical chemistry and clinical medicine, and discloses a steroid hormone marker related to idiopathic male sterility in serum, and a detection method and application thereof. The marker is cortisol and/or dehydroepiandrosterone, is detected by adopting an UPLC-Q active MS method, can be used for auxiliary diagnosis and monitoring of idiopathic male infertility, has high sensitivity and specificity, and has clinical popularization value.
Description
Technical Field
The invention belongs to the fields of analytical chemistry and clinical medicine, and relates to a steroid hormone marker related to idiopathic male sterility in serum, and a detection method and application thereof based on UPLC-Q active MS.
Background
Currently, about 10-15% of women of childbearing age worldwide suffer from fertility disorders. The number of people who are infertile and sterile among newly-married couples in China is far more than million, wherein the male factors cause the sterility to be higher than 50%. Idiopathic male infertility is male infertility for which no clear cause can be found, accounting for 40% -75% of infertility. The WHO diagnosis standard of male infertility is that the couple lives together for more than 1 year after marriage, and no contraceptive measures are taken, so that the male causes the female to be infertile. However, the observation time of the same residence as 1 year greatly delays the time for early treatment and intervention of male infertility; many couples cannot guarantee strict co-existence for 1 year, so that the last judgment of male sterility becomes very difficult; in order to eliminate the reason for the female, the female also needs to perform detailed examination, which brings heavy economic and medical burden. The existing male sterility related examination depends on the conventional semen routine examination, and only focuses on the routine parameters of the number, the vitality, the semen volume, the pH value, the liquefaction time and the like of the sperms. The conventional analysis result of the conventional semen parameters shows larger fluctuation because of the influence of factors such as abstinence time and the like. Thus, clinical diagnosis often requires reference to multiple semen routine analyses. More importantly, conventional semen parameter examination does not fully reflect the entire condition of the semen. Therefore, male infertility is often manifested as no obvious abnormality in conventional semen parameters, and the traditional semen parameter examination cannot effectively diagnose male infertility. Thus, there is a clinical need for new diagnostic methods for idiopathic male infertility.
Metabolomics is an emerging omics technology, which is a science for studying biological systems by high-throughput investigation of their changes in metabolic products following genetic and/or environmental changes. Metabonomics research has the characteristics of unbiased, global, hypothesis-free and data-driven hypothesis, so that the research conclusion is high in reliability and innovation. Metabolomics can systematically review, analyze and explore small molecule biomarkers produced by cells, tissues and organs and changes thereof, and more intuitively understand the material basis of functional phenotype changes, and the objects of the metabolomics relate to metabolic networks of organisms such as lipid metabolism, sugar metabolism, amino acid metabolism, nucleic acid metabolism, coenzyme metabolism and the like, and are called the omics closest to 'phenotype'. Metabonomics shows extremely high application potential and value in the diagnosis of complex diseases, and the analyzed samples can be noninvasive and low-invasive samples such as blood and urine, so that the compliance of patients can be greatly improved, and the metabonomics has the characteristics of high sensitivity and stability. At present, metabonomics is mainly applied to coronary heart disease, liver disease, diabetes, hypertension, obesity and tumor in the aspect of disease diagnosis. Notably, blood is a readily clinically available biological sample with the advantages of low invasiveness and utility for multiple examinations. Is particularly suitable for clinical diagnosis of diseases and screening in physical examination. However, the use of metabolomics for the analysis of small blood metabolic molecules in the diagnostic monitoring of idiopathic male infertility has not received corresponding attention.
The existing common metabonomics detection platforms mainly comprise Nuclear Magnetic Resonance (NMR), gas chromatography tandem mass spectrometry (GC-MS), liquid chromatography tandem mass spectrometry (LC-MS) and capillary electrophoresis tandem mass spectrometry (CE-MS). Although the nuclear magnetic resonance is a gold standard for chemical characterization, the nuclear magnetic resonance has the defect of low sensitivity, and a relatively complete metabolic spectrum is difficult to obtain. Tandem mass spectrometry has the advantages of high sensitivity and accurate characterization. However, gas chromatography tandem mass spectrometry is limited to detecting volatile substances, and the detection range cannot cover the requirement of metabolic spectrum. In order to compensate for the defect, the gas chromatography tandem mass spectrometry often adopts a derivatization method to detect chemicals, which makes the pretreatment method complicated and introduces more errors. Meanwhile, the gas chromatography-tandem mass spectrometry detection is generally longer in detection time due to the limitation of a separation technology, so that the experimental period is prolonged, the instrument occupancy rate is increased, and the application of the gas chromatography-tandem mass spectrometry detection in the detection of a large sample group is limited. Capillary electrophoresis tandem mass spectrometry has advantages in separation and detection of polar compounds, but the problem of instrument stability is difficult to solve. The liquid chromatography tandem mass spectrum has the characteristics of simple sample treatment, high sensitivity and strong clinical practicability, and can well solve the problems. UPLC-Q active MS is a combination of a new generation of high resolution mass spectrum and an ultra-high performance liquid phase, and has higher sensitivity, specificity and stability compared with the traditional LC-MS. Therefore, the UPLC-Q active MS is adopted to carry out metabonomic analysis on the blood metabolism micromolecules, and if stable specific blood metabolism micromolecules related to the onset of the idiopathic male infertility can be found as biomarkers, and a UPLC-Q active MS detection method of the corresponding metabolism micromolecule markers is developed, the UPLC-Q active MS detection method has important clinical application value and can be used for strongly promoting male reproductive health.
Disclosure of Invention
The invention aims to provide a steroid hormone marker related to idiopathic male sterility in blood serum.
The invention also aims to provide a detection method of the steroid hormone marker.
It is still another object of the present invention to provide a kit for detecting the steroid hormone marker.
The aim of the invention is achieved by the following technical measures:
a steroid hormone marker in serum related to idiopathic male infertility, wherein the marker is cortisol and/or dehydroepiandrosterone in serum.
The steroid hormone marker is applied to the preparation of an idiopathic male infertility diagnosis or monitoring reagent.
A kit for diagnosing or monitoring idiopathic male infertility, the kit comprising reagents for detecting serum cortisol and/or dehydroepiandrosterone.
The kit contains a reagent for detecting serum cortisol and/or dehydroepiandrosterone by using a UPLC-Q active MS method.
The kit comprises the following reagents:
cortisol and/or dehydroepiandrosterone standards;
internal standard A: isotope internal standard (deuterium standard, water solution) of one or more substances of creatinine, valine, nicotinic acid, thymine, glutaric acid, L-phenylalanine, N-acetyl-p-aminophenol and hippuric acid;
internal standard B: isotopic internal standards of pentadecanoic acid (deuterium standard, methanol solution);
internal standard C: isotopic internal standard of tetracosanoic acid (deuterium standard, methanol solution).
Further, the kit further comprises:
hypersil GOLD C18 chromatography column;
reagent A: precipitating protein with 100% methanol;
and (3) reagent B: for the mobile phase, water containing 0.1% formic acid;
and (3) reagent C: the mobile phase is acetonitrile containing 0.1 percent of formic acid;
and (3) reagent D: for reconstitution, ultra pure water.
A method for detecting the steroid hormone marker related to the idiopathic male infertility in the serum adopts an UPLC-Q active MS method to detect the content of cortisol and/or dehydroepiandrosterone in the serum.
The detection method comprises the following steps:
firstly, liquid phase conditions:
the liquid chromatographic column is Hypersil GOLD C18 chromatographic column, and the column temperature is 40 ℃;
the mobile phase A is water containing 0.1% formic acid, the mobile phase B is acetonitrile containing 0.1% formic acid, and the flow rate is 400 μ L/min;
the instrument gradient was: 0-3min 1% B, 3-10min 1% to 99% B, 10-13min 99% B, 13-13.1min 99% to 1% B, 13.1-17min 1% B; (B denotes mobile phase B, the amount of mobile phase A in each gradient being 100% of the corresponding amount of mobile phase B, the same applies hereinafter)
And (3) sample introduction mode: volume 5. mu.l;
second, Mass Spectrometry Condition
Analysis was performed using a heating electrospray ionization (HESI) mode, positive ion mode spray voltage: 3.5 kV; negative ion mode spray voltage: 2.5 kV; capillary temperature in two modes: 250 ℃, heater temperature: 425 ℃, sheath gas flow: 50AU, auxiliary gas flow: 13AU, reverse air flow: 0 AU; lens voltage: 60V; adopting a full-scanning mode, wherein the scanning range is as follows: 70 to 1050 m/z; resolution ratio: 70000.
the invention is described in detail below:
the invention collects serum samples meeting the standard by a Standard Operation Program (SOP), systematically collects complete population basic information and clinical data, and adopts a UPLC-Q active MS-based metabonomics method for analysis.
The experimental method of research mainly includes the following parts:
first, research object selection and grouping basis
First stage screening stage
Specifically diagnosed idiopathic male infertility 113 and healthy control 113 were randomized, for a total of 226 people.
Group A: healthy control group (113 people):
1. age between 19 and 38 years;
2. the body mass index is mostly between 17 and 31;
3. a male with healthy reproductive capacity and having healthy offspring after 6-8 months;
4. there is no serious disease of the whole body.
Group B: idiopathic male infertility disease group (113 people):
1. age matched to control group;
2. the body mass index is matched with a control group;
3. a male with no success in attempting pregnancy for 12 months and a spouse without infertility disorder;
4. there is no clear cause of male infertility;
5. the smoking and drinking history is matched with the control group;
6. matching nationality with a control group;
7. there is no serious disease of the whole body.
Second phase verification phase
Specifically diagnosed idiopathic male infertility 15 and healthy controls 15 were included, for a total of 30.
Group A: healthy control group (15 persons):
1. age between 24 and 36 years;
2. a body mass index between 19 and 24;
3. a male with healthy reproductive capacity and having healthy offspring after 6-8 months;
4. there is no serious disease of the whole body.
Group B: idiopathic male infertility disease group (15 people):
1. age matched to control group;
2. the body mass index is matched with a control group;
3. a male with no success in attempting pregnancy for 12 months and a spouse without infertility disorder;
4. there is no clear cause of male infertility;
5. the smoking and drinking history is matched with the control group;
6. matching nationality with a control group;
7. there is no serious disease of the whole body.
II, screening and verification of serum biomarkers cortisol and dehydroepiandrosterone for UPLC-Q active MS metabonomics analysis and idiopathic male sterility diagnosis
1. Sample pretreatment
1.1. Add 10. mu.L of serum to 10. mu.L of internal standard A, 10. mu.L of internal standard B, 10. mu.L of internal standard C, 40. mu.L of methanol (reagent A) and vortex for 30 s.
1.2. The supernatant was transferred to a 1.5mL inlet EP tube after centrifugation at 16000g in a centrifuge at 4 ℃ for 15min and concentrated to dryness in a centrifugal concentration desiccator at room temperature.
1.3. Reconstituted with 5. mu.L of ultrapure water (reagent D) and ready for analysis.
2. Instrumental detection
2.1. An analytical instrument: UPLC Ultimate 3000system (dionex) hplc; q-active high resolution mass spectrometer.
2.2. Liquid phase conditions:
2.2.1. the liquid chromatography column was a Hypersil GOLD C18 column (100 mm. times.2.1 mm, particle size 1.9 μm, Thermo Scientific, Germany) at a column temperature of 40 ℃.
2.2.2 the mobile phase A used was water containing 0.1% formic acid (reagent B) and the mobile phase B was acetonitrile containing 0.1% formic acid (reagent C) at a flow rate of 400. mu.L/min.
2.2.3 Instrument gradient: 0-3min 1% B, 3-10min 1% to 99% B, 10-13min 99% B, 13-13.1min 99% to 1% B, 13.1-17min 1% B.
2.2.4 sample introduction mode: volume 5. mu.l.
2.3. Conditions of Mass Spectrometry
2.3.1 analysis by heated electrospray ionization (HESI).
2.3.2 using the heating electrospray ionization mode (HESI), positive ion mode spray voltage: 3.5 kV; negative ion mode spray voltage: 2.5 kV; capillary temperature in two modes: 250 ℃, heater temperature: 425 ℃, sheath gas flow: 50AU, auxiliary gas flow: 13AU, reverse air flow: 0 AU; lens voltage: 60V. Adopting a full-scanning mode, wherein the scanning range is as follows: 70 to 1050 m/z; resolution ratio: 70000.
3. characterization of the substance
The biomarker characterization adopts the comparison of chromatographic information (retention time) and mass spectrum information (accurate molecular weight) with standard cortisol and dehydroepiandrosterone, and compares the chromatographic information of isotope internal standard series in a sample in real time to correct the retention time.
4. And (3) data analysis:
biomarker screening key metabolites were confirmed using multiple Logistic regression.
5. The difference and the diagnosis significance of cortisol and dehydroepiandrosterone in the serum of a healthy control group and an idiopathic male sterility group.
After correcting the information of age, body mass index, smoking and drinking history, multivariate Logistic regression analysis finds that the contents of cortisol and dehydroepiandrosterone in a serum sample are closely related to idiopathic male infertility. The metabolic small molecule combination is applied to random population to diagnose idiopathic male infertility, the sensitivity is 86.67%, the specificity is 93.33%, the area under an ROC curve is 0.9244, and the method has high auxiliary diagnosis value.
Third, preparation method of diagnostic kit
According to the series of experimental results, the inventor also prepares a kit capable of diagnosing or monitoring idiopathic male infertility, wherein the kit contains reagents for detecting serum biomarkers cortisol and dehydroepiandrosterone related to the idiopathic male infertility, and the kit comprises standards for measuring stably existing and detectable cortisol and dehydroepiandrosterone in the serum of a subject and an internal standard series for auxiliary analysis. The diagnostic kit can also comprise a set of reagents and devices for extracting serum cortisol and dehydroepiandrosterone and performing chromatographic separation.
The invention has the beneficial effects that:
the inventor discovers a serum cortisol and dehydroepiandrosterone combination which can be used for evaluating whether the idiopathic male sterility is suffered and has diagnostic value and the application of the UPLC-Q active MS for detecting the serum cortisol and the dehydroepiandrosterone by comparing the metabolic small molecules in the serum of a normal control and the idiopathic male sterility by using the UPLC-Q active MS, and develops the idiopathic male sterility auxiliary diagnosis and monitoring kit which can be conveniently applied clinically.
The invention adopts serum metabolism micromolecules as markers for evaluating idiopathic male sterility, and has the advantages that:
(1) the serum metabolism micromolecule is a novel biomarker which is strongly associated with disease fate, is stable, minimally invasive, easy to detect and accurate in quantification, can greatly improve the sensitivity and specificity of idiopathic male infertility diagnosis, and the successful development of the micromolecule biomarker can create a brand new situation for the prevention and treatment of the idiopathic male infertility and provide reference for the development of other disease biomarkers.
(2) The serum metabolism micromolecule marker provided by the invention can be used as a diagnostic marker of idiopathic male infertility, and can assist in diagnosing the idiopathic male infertility at an early stage, thereby providing a basis for further in-depth examination of a clinician, providing support for rapidly and accurately mastering the disease state and the disease severity of a patient, timely adopting a more personalized prevention and treatment scheme, and delaying and stopping disease progression.
(3) The invention adopts serum samples of idiopathic male infertility and healthy control random population for verification, proves that the cortisol and dehydroepiandrosterone level in the serum has higher sensitivity and specificity in diagnosing the idiopathic male infertility, and can be used as a marker.
(4) The invention adopts a strict and multistage verification and evaluation system, screens a plurality of serum metabolism micromolecules through preliminary experiments at the initial stage, and uses UPLC-Q active MS to verify independent population, thereby ensuring the reliability of the serum metabolism biomarker and the diagnosis method.
(5) The UPLC-Q active MS technology has the advantages of simple sample processing, rapid and accurate instrument analysis and higher clinical diagnosis practical value.
Drawings
Figure 1 shows that serum cortisol and dehydroepiandrosterone are closely related to idiopathic male infertility in a screening stage through information of corrected age, body mass index, smoking and drinking history and multivariate Logistic regression analysis.aThe one-way Logistic regression results for confounding factors were not adjusted.bAdjusting multiple Logistic regression results after age, body mass index, smoking and drinking history.
FIG. 2 Metabolic assay level variability (mean. + -. standard deviation).
FIG. 3 shows ROC curve between normal control group and idiopathic male sterility group prepared by using serum cortisol content information in validation stage.
FIG. 4 shows ROC curve between normal control group and idiopathic male sterility group prepared by using serum dehydroepiandrosterone content information in verification stage.
FIG. 5 shows the ROC curve between the normal control group and the idiopathic male sterility group prepared by the information of the contents of serum cortisol and dehydroepiandrosterone in the verification stage.
Detailed Description
The invention is further illustrated by the following examples.
Example 1: subject selection and grouping basis
The study subjects were from the first-visit idiopathic male infertility case and healthy fertility control of the subsidiary hospital of the Nanjing medical university. The research content and the informed consent were approved by the ethical committee of the university of medical Nanjing, and were in compliance with the requirements of the relevant regulations. Cases and controls signed informed consent after understanding the content. All subjects performed a complete physical examination and completed a questionnaire that included personal basic data, lifestyle habits, occupational and environmental exposure, genetic risk factors, sexual and reproductive function, disease history, and physical activity. In the first stage, 113 cases of idiopathic male infertility and 113 healthy controls were included as required; 15 cases of idiopathic male infertility and 15 healthy controls meeting the requirements of the second stage were used as screening subjects of serum biomarkers of idiopathic male infertility. The specific sample classification criteria are as follows:
first stage screening stage
Specifically diagnosed idiopathic male infertility 113 and healthy control 113 were randomized, for a total of 226 people.
Group A: healthy control group (113 people):
1. age between 19 and 38 years;
2. the body mass index is mostly between 17 and 31;
3. a male with healthy reproductive capacity and having healthy offspring after 6-8 months;
4. there is no serious disease of the whole body.
Group B: idiopathic male infertility disease group (113 people):
1. age matched to control group;
2. the body mass index is matched with a control group;
3. a male with no success in attempting pregnancy for 12 months and a spouse without infertility disorder;
4. there is no clear cause of male infertility;
5. the smoking and drinking history is matched with the control group;
6. matching nationality with a control group;
7. there is no serious disease of the whole body.
Second phase verification phase
Specifically diagnosed idiopathic male infertility 15 and healthy controls 15 were included, for a total of 30.
Group A: healthy control group (15 persons):
1. age between 24 and 36 years;
2. a body mass index between 19 and 24;
3. a male with healthy reproductive capacity and having healthy offspring after 6-8 months;
4. there is no serious disease of the whole body.
Group B: idiopathic male infertility disease group (15 people):
1. age matched to control group;
2. the body mass index is matched with a control group;
3. a male with no success in attempting pregnancy for 12 months and a spouse without infertility disorder;
4. there is no clear cause of male infertility;
5. the smoking and drinking history is matched with the control group;
6. matching nationality with a control group;
7. there is no serious disease of the whole body.
Example 2: UPLC-MS metabonomics idiopathic male sterility biomarker screening
1. Sample pretreatment
1.1. mu.L of serum was taken, 10. mu.L of internal standard A was added, 10. mu.L of internal standard B was added, 10. mu.L of internal standard C was added, 40. mu.L of methanol (reagent A) was added, and vortexed for 30 s.
1.2. The supernatant was transferred to a 1.5mL inlet EP tube after centrifugation at 16000g in a centrifuge at 4 ℃ for 15min and concentrated to dryness in a centrifugal concentration desiccator at room temperature.
1.3. Reconstituted with 5. mu.L of ultrapure water (reagent D) and ready for analysis.
2. Instrumental detection
2.1. An analytical instrument: UPLC Ultimate 3000system (dionex) hplc; q-active high resolution mass spectrometer.
2.2. Liquid phase conditions:
2.2.1 the liquid chromatography column was a Hypersil GOLD C18 column (100 mm. times.2.1 mm, particle size 1.9 μm, Thermo Scientific, Germany) at a column temperature of 40 ℃.
2.2.2 the mobile phases used were (A) water containing 0.1% formic acid (reagent B) and (B) acetonitrile containing 0.1% formic acid (reagent C) at a flow rate of 400. mu.L/min.
2.2.3 Instrument gradient: 0-3min 1% B, 3-10min 1% to 99% B, 10-13min 99% B, 13-13.1min 99% to 1% B, 13.1-17min 1% B.
2.2.4 sample introduction mode: volume 5. mu.l.
2.3. Conditions of Mass Spectrometry
2.3.1 analysis by heated electrospray ionization (HESI).
2.3.2 using the heating electrospray ionization mode (HESI), positive ion mode spray voltage: 3.5 kV; negative ion mode spray voltage: 2.5 kV; capillary temperature in two modes: 250 ℃, heater temperature: 425 ℃, sheath gas flow: 50AU, auxiliary gas flow: 13AU, reverse air flow: 0 AU; lens voltage: 60V. Adopting a full-scanning mode, wherein the scanning range is as follows: 70 to 1050 m/z; resolution ratio: 70000.
3. characterization of the substance
The biomarker characterization adopts the comparison of chromatographic information (retention time) and mass spectrum information (accurate molecular weight) with standard cortisol and dehydroepiandrosterone, and compares the chromatographic information of isotope internal standard series in a sample in real time to correct the retention time.
4. And (3) data analysis:
biomarker screening key metabolites were confirmed using multiple Logistic regression.
5. The difference and the diagnostic significance of cortisol and dehydroepiandrosterone in serum samples of a healthy control group and an idiopathic male sterility group.
After correcting the information of age, body mass index, smoking and drinking history, multivariate Logistic regression analysis finds that serum cortisol and dehydroepiandrosterone are closely related to idiopathic male infertility (figure 1).
Example 3 stability analysis of Cortisol and dehydroepiandrosterone in serum
The stability of cortisol and dehydroepiandrosterone levels in serum was evaluated using the method of example 2 (interval 2 weeks). The results show that serum cortisol and dehydroepiandrosterone levels were stable (fig. 2), and were characteristic as diagnostic/monitoring markers.
Example 4 diagnosis of idiopathic male infertility by combination of Cortisol and dehydroepiandrosterone in serum
According to the UPLC-Q active MS metabonomics method, the inventor detects cortisol and dehydroepiandrosterone by serum samples of 15 random population cases and 15 control cases, draws an ROC curve and evaluates the sensitivity and specificity of diagnosis, and further evaluates the diagnostic capability of detecting the 2 substance levels in the serum on idiopathic male infertility.
The sensitivity of cortisol was 86.67%, the specificity was 86.67%, and the area under the ROC curve was 0.8933 (fig. 3); dehydroepiandrosterone sensitivity was 73.33%, specificity was 80.00%, and area under the ROC curve was 0.8089 (FIG. 4).
The sensitivity of the combination cortisol and dehydroepiandrosterone was 86.67%, the specificity was 93.33%, and the area under the ROC curve was 0.9244 (fig. 5).
Therefore, the combination of cortisol and dehydroepiandrosterone has better capability of diagnosing idiopathic male infertility.
EXAMPLE 5 preparation of kit for detecting and diagnosing cortisol and dehydroepiandrosterone in idiopathic male sterility serum
Firstly, determining metabolic small molecules with higher abundance in serum of normal control and idiopathic male sterility by using a UPLC-Q active MS method. Then, biomarkers related to the idiopathic male sterility are screened by the UPLC-Q active MS-based metabonomics technology as a diagnostic index of whether the idiopathic male sterility is detected. The number of corresponding serum biomarkers screened is preferably controlled to 2, which is a reduction of the optimization made on a preliminary experiment basis. The 2 biomarkers of cortisol and dehydroepiandrosterone in serum are adopted, so that the method can ensure better sensitivity and specificity, save cost, reduce the burden of a patient, reduce the detection time, has the advantages of rapidness, accuracy and economy, is convenient for clinical popularization and use, and can also adopt 1 marker in the serum, and has better effect by adopting 2 markers. This kit includes reagent and consumptive material for a batch serum cortisol and dehydroepiandrosterone detection, and wherein cortisol and dehydroepiandrosterone standard substance are adopted to the qualitative and ration of biomarker, and the auxiliary analysis adopts interior mark A: the deuterium labeled isotope internal standard of eight substances of creatinine, valine, nicotinic acid, thymine, glutaric acid, L-phenylalanine, N-acetyl-p-aminophenol and hippuric acid. Internal standard B: deuterium-labelled isotopic internal standard of pentadecanoic acid. Internal standard C: deuterium-labelled isotopic internal standard of tetracosanoic acid. Other examples are a reversed phase chromatographic column (Hypersil GOLD C18 column, 100 mm. times.2.1 mm, particle size 1.9 μm), a reagent for precipitating serum proteins (100% methanol), a reagent for mobile phase (0.1% formic acid in water and 0.1% formic acid in acetonitrile), and a reagent for extraction of cortisol and dehydroepiandrosterone (100% ultrapure water) for UPLC chromatographic separation. The kit has the value that the content of cortisol and dehydroepiandrosterone markers in serum can be detected only by 10 mul of serum, then idiopathic male infertility is diagnosed through the content, and dynamic monitoring and treatment effect observation are easy to carry out.
The specific kit comprises the following components:
cortisol standard substance
Dehydroepiandrosterone standard substance
Internal standard A (deuterium isotope internal standard water solution of eight substances of creatinine, valine, nicotinic acid, thymine, glutaric acid, L-phenylalanine, N-acetyl-p-aminophenol and hippuric acid)
Internal standard B (methanol solution with deuterium standard isotope of pentadecanoic acid)
Internal standard C (methanol solution with deuterium isotope internal standard of tetracosanoic acid)
Further, the method may further comprise:
chromatographic column (Thermo 100mm X2.1 mm, particle size 1.9 μm, Hypersil GOLD C18 chromatographic column)
Reagent A (100% methanol)
Reagent B (Water containing 0.1% formic acid)
Reagent C (acetonitrile containing 0.1% formic acid)
Reagent D (100% ultrapure water).
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1. The application of a steroid hormone marker related to idiopathic male sterility in serum in preparing a diagnostic or monitoring reagent for idiopathic male sterility, wherein the marker is cortisol and dehydroepiandrosterone.
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