CN106568852A - Idiopathic male infertility related steroid hormone marker in serum, detection method and application thereof - Google Patents
Idiopathic male infertility related steroid hormone marker in serum, detection method and application thereof Download PDFInfo
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Abstract
Belonging to the field of analytical chemistry and clinical medicine, the invention discloses an idiopathic male infertility related steroid hormone marker in serum, a detection method and application thereof. The marker is cortisol and/or dehydroepiandrosterone, a UPLC-Q exactive MS method is employed for detection, the marker can be used for auxiliary diagnosis and monitoring of idiopathic male infertility, has high sensitivity and specificity, and has clinical promotion value.
Description
Invention field
The invention belongs to analytical chemistry and clinical medicine domain, are related to class related to idiopathic male infertility in serum and consolidate
Alcohol hormone mark and its detection method based on UPLC-Q exactive MS and application.
Background technology
At present, the whole world there are about the couple at child-bearing age of 10-15% with growing barrier.Infertile people in China newly-married couple
Number exceeds well over million, and infertility is higher than 50% wherein caused by male factor.Idiopathic male infertility disease is to can not find the clear and definite cause of disease
Male sterility, account for infertility 40%-75%.The diagnostic criteria of the WHO of male sterility is that man and wife lives together after marriage more than 1 year, not
Contraceptives are taken, because bridegroom's or husband's side reason causes the infertile person in the wife's side.However, living together up to the observing time of 1 year, delay significantly
Early treatment and the time intervened are carried out to male sterility；Many men and wives do not ensure that strict living together 1 year so that last
Male sterility judges to become extremely difficult；In order to exclude wife's side reason, the wife's side also needs to carry out detailed inspection, brings heavy
Economy and medical burden.What existing male sterility coherence check was relied on is traditional Sperm recovery, and it only focuses on essence
The conventional parameters such as quantum count, vigor, semen volume, pH and liquefying time.Because being affected by factors such as abstinence times, conventional essence
Liquid parameter conventional analysis result shows as larger fluctuation.Thus, clinical diagnosis is generally required with reference to multiple Semen routione point
Analysis.What is more important, traditional seminal parameters inspection can not comprehensively react whole situations of seminal fluid.Therefore, male sterility
Jing often shows as Sperm parameters Non Apparent Abnormality, also cause traditional seminal parameters inspection can not the efficient diagnosis male sex not
Educate.Thus, clinically need the new diagnostic method for idiopathic male infertility badly.
Metabolism group is emerging omics technology, and it is to investigate living things system in hereditary change and/or ring by high flux
The a science of the Changeement living things system of metabolite after the change of border.Metabolism group research have " unbiased ", " overall situation ",
The characteristics of " without assuming ", " being assumed by data-driven ", thus the conclusion of research often has higher reliability and novelty.Generation
Small molecule biomarkers thing and its change that Xie Zuxue can be produced with systematic overview, analysis and exploration cell, tissue and organ
Situation, is more visually known the material base of function phenotypic alternation, its object be related to lipid metaboli, glycometabolism, amino acid metabolism,
The metabolism network of the organisms such as nucleic acid metabolism, coenzyme metabolism, is referred to as the group closest to " phenotype ".Metabolism group is in complicated disease
High application potential and value are shown in the diagnosis of disease, the sample analyzed due to it can for blood urine these it is noninvasive and
The sample of low wound, is greatly improved the compliance of patient, and the characteristics of with sensitivity high stable.At present metabolism group is in disease
Diagnosis aspect be mainly used in coronary heart disease, liver diseases, diabetes, hypertension, obesity and tumour.It should be noted that blood
Liquid is the biological specimen being clinically readily available, and has the advantages that low wound and multiple inspection are public.It is particularly suitable for the clinic of disease
Examination in diagnosis and health check-up.However, using metabonomic analysis plasma metabolism small molecule idiopathic male infertility diagnosis
Application in monitoring is also paid close attention to accordingly.
Existing common metabolism group detection platform mainly includes nuclear magnetic resonance (NMR), gas-chromatography tandem mass spectrum (GC-
MS), Liquid Chromatography-Tandem Mass Spectrometry (LC-MS) and Capillary Electrophoresis tandem mass spectrum (CE-MS).Although nuclear magnetic resonance is that chemicals are determined
Property goldstandard, but have the shortcomings that sensitivity is low, it is difficult to obtain more complete metabolism spectrum.Tandem mass spectrum has sensitivity high
With qualitative accurate advantage.But, gas-chromatography tandem mass spectrum is only limited to detect volatile material that detection range can not be covered
The requirement of metabolism spectrum.In order to make up the defect, gas-chromatography tandem mass spectrum often detects chemicals using the method for derivatization, this
So that pre-treating method becomes complicated, more errors are introduced.Meanwhile, gas-chromatography tandem mass spectrum is detected due to isolation technics
Limit, general detection time is longer, extends experimental period, increased instrument occupancy, limit it and examine in large sample crowd
Application in survey.Capillary Electrophoresis tandem mass spectrum, have advantage to the separation detection of polar compound, but stability of instrument is asked
Topic is relatively difficult to resolve certainly.And Liquid Chromatography-Tandem Mass Spectrometry has sample process simple, sensitivity is high, the characteristics of Clinical practicability is strong, can
To solve the above problems well.UPLC-Q exactive MS are the combinations of high resolution mass spectrum of new generation and ultra high efficiency liquid phase,
With comparing the higher sensitivity of traditional LC-MS, specificity and stability.So promoting circulation of blood is entered using UPLC-Q exactive MS
The metabonomic analysis of liquid metabolism small molecule, if the stable special blood generation related to idiopathic male infertility morbidity can be found
Thank to small molecule as biomarker, and research and develop the UPLC-Q exactive MS detection sides of corresponding metabolism small molecule mark
Method, by with important clinical value, strong promotion is made to male reproductive health.
The content of the invention
It is an object of the invention to provide steroid hormone mark related to idiopathic male infertility in serum.
Another object of the present invention is to provide the detection method of above-mentioned steroid hormone mark.
A further object of the present invention is to provide the kit for detecting above-mentioned steroid hormone mark.
The purpose of the present invention is realized by following technical measures：
Steroid hormone mark related to idiopathic male infertility in serum, the mark is the cortisol in serum
And/or dehydrobenzene.
Described steroid hormone mark is preparing idiopathic male infertility diagnosis or the application in monitoring reagent.
It is a kind of diagnosis or monitor idiopathic male infertility kit, the kit contain detection serum cortisol and/or
The reagent of dehydrobenzene.
Described kit, the kit contain using UPLC-Q exactive MS methods detection serum cortisol and/
Or the reagent of dehydrobenzene.
Described kit, the kit contains following reagent：
Cortisol and/or dehydrobenzene standard items；
Internal standard A：Creatinine, valine, nicotinic acid, thymidine, glutaric acid, the acid of L- phenylpropylamines, N- acetaminophens, horse
The Isotopic Internal Standard (deuterium mark, the aqueous solution) of one or more material in uric acid；
Internal standard B：The Isotopic Internal Standard (deuterium mark, methanol solution) of pentadecanoic acid；
Internal standard C：The Isotopic Internal Standard (deuterium mark, methanol solution) of lignoceric acid.
Further, the kit also contains：
Hypersil GOLD C18 chromatographic columns；
Reagent A：Protein precipitation is used, containing 100% methyl alcohol；
Reagent B：Mobile phase is used, the water containing 0.1% formic acid；
Reagent C：Mobile phase is used, the acetonitrile containing 0.1% formic acid；
Reagent D：Redissolve and use, ultra-pure water.
A kind of method for detecting steroid hormone mark related to idiopathic male infertility in foregoing serum,
The method adopts UPLC-Q exactive MS methods, cortisol and/or dehydrobenzene content in detection serum.
In above-mentioned detection method：
First, liquid-phase condition：
Liquid-phase chromatographic column is Hypersil GOLD C18 chromatographic columns, and column temperature is 40 DEG C；
Mobile phase A is the water containing 0.1% formic acid, and Mobile phase B is the acetonitrile containing 0.1% formic acid, and flow velocity is 400 μ L/min；
Instrument gradient is：0-3min 1%B, 3-10min 1% arrive 99%B, 10-13min 99%B, 13-13.1min
99% arrives 1%B, 13.1-17min 1%B；(B refers to Mobile phase B, the amount of the amount of mobile phase A and corresponding Mobile phase B in each gradient
Totally 100%, similarly hereinafter)
Input mode：The μ l of volume 5；
2nd, Mass Spectrometry Conditions
It is analyzed using heating electron spray ionisation mode (HESI), positive ion mode spray voltage：3.5kV；Anion
Pattern spray voltage：2.5kV；Capillary temperature under both of which：250 DEG C, heter temperature：425 DEG C, sheath gas air-flow：50AU,
Auxiliary gas air-flow：13AU, blowback air air-flow：0AU；Lens voltage：60V；Using sweeping pattern, sweep limits entirely：70 arrive 1050m/
z；Resolution ratio：70000.
The present invention is described in detail as follows：
The present inventor gathers standard compliant serum sample, the complete crowd of systematic collection with S.O.P. (SOP)
Back ground Information and clinical data, and employ and be analyzed based on the metabolism group method of UPLC-Q exactive MS.
The experimental technique specifically studied mainly includes following components：
First, research object selects and is grouped foundation
First stage screening stage
The people of idiopathic male infertility 113 and the people of normal healthy controls 113 for clarifying a diagnosis is included at random, totally 226 people.
A groups：Healthy control group (113 people)：
1. the age is between 19 to 38 years old；
2. constitutional index major part is between 17 to 31；
3. healthy offspring is had after the male sex of fecundity health, and 6-8 month；
4. without whole body major disease.
B groups：Idiopathic male infertility disease group (113 people)：
1. the age match with control group；
2. constitutional index is matched with control group；
3. pregnancy is attempted 12 months without success, and spouse does not have the male sex of infertile disease；
4. without the clear and definite male sterility cause of disease；
5. Smoking And Drinking history is matched with control group；
6. nationality matches with control group；
7. without whole body major disease.
Second stage Qualify Phase
The people of idiopathic male infertility 15 and the people of normal healthy controls 15 for clarifying a diagnosis is included, totally 30 people.
A groups：Healthy control group (15 people)：
1. the age is between 24 to 36 years old；
2. constitutional index is between 19 to 24；
3. healthy offspring is had after the male sex of fecundity health, and 6-8 month；
4. without whole body major disease.
B groups：Idiopathic male infertility disease group (15 people)：
1. the age match with control group；
2. constitutional index is matched with control group；
3. pregnancy is attempted 12 months without success, and spouse does not have the male sex of infertile disease；
4. without the clear and definite male sterility cause of disease；
5. Smoking And Drinking history is matched with control group；
6. nationality matches with control group；
7. without whole body major disease.
2nd, UPLC-Q exactive MS metabonomic analysis and idiopathic male infertility diagnosis serum biomarkers
The screening and checking of cortisol and dehydrobenzene
1. Sample pretreatment
1.1. 10 μ L serum are taken, 10 μ L internal standards A are added, 10 μ L internal standards B are added, 10 μ L internal standards C are added, the μ L of methyl alcohol 40 are added
(reagent A), vortex 30s.
1.2. 4 DEG C of 16000g are centrifuged 15min in centrifuge, and supernatant is transferred to into 1.5mL imports EP pipes, and by supernatant
It is concentrated to dryness in centrifugal concentrating drying instrument at ambient temperature.
1.3. redissolved with 5 μ L ultra-pure waters (reagent D), it is to be analyzed.
2. instrument detection
2.1. analytical instrument：UPLC Ultimate 3000system (Dionex) high performance liquid chromatographs；Q-
Exactive high-resolution mass spectrometers.
2.2. liquid-phase condition：
2.2.1. liquid-phase chromatographic column be Hypersil GOLD C18 chromatographic columns (100mm × 2.1mm, 1.9 μm of particle diameter,
Thermo Scientific, Germany), column temperature is 40 DEG C.
2.2.2 the mobile phase A for adopting is the water (reagent B) containing 0.1% formic acid and Mobile phase B is the second containing 0.1% formic acid
Nitrile (reagent C), flow velocity is 400 μ L/min.
2.2.3 instrument gradient is：0-3min 1%B, 3-10min 1% arrive 99%B, 10-13min 99%B, 13-
13.1min99% to 1%B, 13.1-17min 1%B.
2.2.4 input mode：The μ l of volume 5.
2.3. Mass Spectrometry Conditions
2.3.1 heat electron spray ionisation mode (HESI) to be analyzed.
2.3.2 using heating electron spray ionisation mode (HESI), positive ion mode spray voltage：3.5kV；Negative ion mode
Spray voltage：2.5kV；Capillary temperature under both of which：250 DEG C, heter temperature：425 DEG C, sheath gas air-flow：50AU, auxiliary
Gas air-flow：13AU, blowback air air-flow：0AU；Lens voltage：60V.Using sweeping pattern, sweep limits entirely：70 arrive 1050m/z；Point
Resolution：70000.
3. material is qualitative
Biomarker it is qualitative using with standard items cortisol and dehydrobenzene compare Chromatographic information (retention time) and
Information in Mass Spectra (accurate molecular weight), and the Chromatographic information of Isotopic Internal Standard standard items series in sample is compared in real time to correct reservation
Time.
4. data analysis：
Biomarker screening confirms key metabolites using Multivariate Logistic Regression.
5. the difference and diagnosis of cortisol and dehydrobenzene are anticipated in healthy control group, idiopathic male infertility group serum
Justice.
The information of corrected age, constitutional index, smoking and history of drinking history, logistic regression finds serum
The content of cortisol and dehydrobenzene is closely related with idiopathic male infertility in sample.In above-mentioned generation, is applied using random crowd
Thank to small molecule combinatorial diagnosis idiopathic male infertility, sensitivity is 86.67%, and specificity is 93.33%, area under ROC curve
For 0.9244, with higher additive diagnostic value.
3rd, diagnostic reagent box preparation method
According to above-mentioned series of experiments result, the present inventor is also prepared for one kind and can diagnose or monitor idiopathic male infertility
Kit, the kit contains for detecting the serum biomarkers cortisol related to idiopathic male infertility and taking off
The reagent of hydrogen meter androsterone, comprising the standard for determining stable existence and detectable cortisol and dehydrobenzene in experimenter's serum
The internal standard standard items series of product and assistant analysis.Diagnostic kit can also include a set of serum cortisol and dehydrobenzene
Extract and used in chromatograph reagent and equipment.
Beneficial effects of the present invention：
The present inventor using UPLC-Q exactive MS by being compared in normal control and idiopathic male infertility serum
Metabolism small molecule, it was found that exist in serum and can be used to assessing whether with idiopathic male infertility, with diagnostic value
Serum cortisol and dehydrobenzene are combined, and the serum cortisol and the UPLC-Q exactive of dehydrobenzene detection
The application of MS, developing can be easy to idiopathic male infertility auxiliary diagnosis, the monitoring reagent box of clinical practice.
The present invention is advantageous in that using blood serum metabolic small molecule as the mark of idiopathic male infertility evaluation：
(1) blood serum metabolic small molecule is a kind of new biomarkers, and it is associated by force with disease outcome, not only stable, micro-
Wound, it is easy to detection, and quantitative accurate, will greatly improves the Sensitivity and Specificity of idiopathic male infertility diagnosis, such little point
The successful exploitation of sub- biomarker will start brand-new situation for the preventing and treating of idiopathic male infertility, be other diseases biological marker
The development of thing is offered reference.
(2) the blood serum metabolic small molecule mark that the present invention is provided can be used as the diagnosis marker of idiopathic male infertility,
, so as to provide foundation for the further testing in depth testing of clinician, can be quick standard in early stage auxiliary diagnosis idiopathic male infertility
The morbid state and coincident with severity degree of condition of really grasping patient, the control prece for taking more personalized in time provide support, delay
With prevention progression of disease.
(3) present invention is verified using the serum sample of idiopathic male infertility and the random crowd of normal healthy controls, it was demonstrated that
Cortisol and dehydroepiandrosterolevels levels have higher sensitivity and specificity in diagnosis idiopathic male infertility in serum, can
Use as mark.
(4) using tight, multistage checking and appraisement system, the initial stage screens various serum generations to the present invention by preliminary experiment
Thank to small molecule, using UPLC-Q exactive MS independent crowd's checking carried out, it is ensured that the blood serum metabolism biological marker and
The reliability of diagnostic method.
(5) UPLC-Q exactive MS technologies sample process is simple, and Instrumental Analysis is accurate rapidly, with higher clinic
Diagnosis practical value.
Description of the drawings
Fig. 1. screening stage, the information of corrected age, constitutional index, smoking and history of drinking history, polynary Logistic is returned
Analysis is returned to find that serum cortisol and dehydrobenzene are closely related with idiopathic male infertility.aThe list of Confounding Factor is not adjusted
Factor Logistic regression result.bMultivariate Logistic Regression result after adjustment age, constitutional index, smoking and history of drinking history.
Fig. 2. metabolism detection level fluctuation (mean ± standard deviation).
Fig. 3. Qualify Phase, the Normal group made using serum cortisol content information and idiopathic male infertility group
Between ROC curve.
Fig. 4. Qualify Phase, the Normal group made using serum dehydroepiandrosterolevels content information and the idiopathic male sex are not
Educate the ROC curve between group.
Fig. 5. Qualify Phase, the Normal group made using serum cortisol and dehydrobenzene content information and Te Fa
ROC curve between property male sterility group.
Specific embodiment
By the following examples the invention will be further elaborated.
Embodiment 1：Research object selects and is grouped foundation
This part research object examines idiopathic male infertility case and health from the head of affiliated hospital of Nanjing Medical University
Fertility control.Research contents and Informed Consent Form obtain the approval of Ethics Committee of Nanjing Medical University, meet relevant laws and regulations
Requirement.Case and to impinge upon understand content after endorsed Informed Consent Form.All research objects have carried out complete physique
Check, and complete a including personal basic data, habits and customs, occupation and environmental exposure, genetic risk factors, sexual function
With the questionnaire of reproductive function, history of disease and physical exertion.First stage incorporates satisfactory 113 idiopathic male sex
Sterile case and 113 normal healthy controls；The satisfactory 15 idiopathic male infertility cases of second stage and 15 health are right
According to the screening experiment object as idiopathic male infertility serum biomarkers.Specific sample group standard is as follows：
First stage screening stage
The people of idiopathic male infertility 113 and the people of normal healthy controls 113 for clarifying a diagnosis is included at random, totally 226 people.
A groups：Healthy control group (113 people)：
1. the age is between 19 to 38 years old；
2. constitutional index major part is between 17 to 31；
3. healthy offspring is had after the male sex of fecundity health, and 6-8 month；
4. without whole body major disease.
B groups：Idiopathic male infertility disease group (113 people)：
1. the age match with control group；
2. constitutional index is matched with control group；
3. pregnancy is attempted 12 months without success, and spouse does not have the male sex of infertile disease；
4. without the clear and definite male sterility cause of disease；
5. Smoking And Drinking history is matched with control group；
6. nationality matches with control group；
7. without whole body major disease.
Second stage Qualify Phase
The people of idiopathic male infertility 15 and the people of normal healthy controls 15 for clarifying a diagnosis is included, totally 30 people.
A groups：Healthy control group (15 people)：
1. the age is between 24 to 36 years old；
2. constitutional index is between 19 to 24；
3. healthy offspring is had after the male sex of fecundity health, and 6-8 month；
4. without whole body major disease.
B groups：Idiopathic male infertility disease group (15 people)：
1. the age match with control group；
2. constitutional index is matched with control group；
3. pregnancy is attempted 12 months without success, and spouse does not have the male sex of infertile disease；
4. without the clear and definite male sterility cause of disease；
5. Smoking And Drinking history is matched with control group；
6. nationality matches with control group；
7. without whole body major disease.
Embodiment 2：UPLC-MS metabolism group idiopathic male infertilities biomarker is screened
1. Sample pretreatment
1.1. 10 μ L serum are taken, 10 μ L internal standards A are added, 10 μ L internal standards B are added, 10 μ L internal standards C are added, methyl alcohol (examination is added
Agent A) 40 μ L, vortex 30s.
1.2. 4 DEG C of 16000g are centrifuged 15min in centrifuge, and supernatant is transferred to into 1.5mL imports EP pipes, and by supernatant
It is concentrated to dryness in centrifugal concentrating drying instrument at ambient temperature.
1.3. redissolved with 5 μ L ultra-pure waters (reagent D), it is to be analyzed.
2. instrument detection
2.1. analytical instrument：System (Dionex) high performance liquid chromatographs of UPLC Ultimate 3000；Q-
Exactive high-resolution mass spectrometers.
2.2. liquid-phase condition：
2.2.1 liquid-phase chromatographic column be Hypersil GOLD C18 chromatographic columns (100mm × 2.1mm, 1.9 μm of particle diameter,
Thermo Scientific, Germany), column temperature is 40 DEG C.
2.2.2 the mobile phase for adopting is that the acetonitrile of the water (reagent B) of (A) containing 0.1% formic acid and (B) containing 0.1% formic acid (is tried
Agent C), flow velocity is 400 μ L/min.
2.2.3 instrument gradient is：0-3min 1%B, 3-10min 1% arrive 99%B, 10-13min 99%B, 13-
13.1min99% to 1%B, 13.1-17min 1%B.
2.2.4 input mode：The μ l of volume 5.
2.3. Mass Spectrometry Conditions
2.3.1 heat electron spray ionisation mode (HESI) to be analyzed.
2.3.2 using heating electron spray ionisation mode (HESI), positive ion mode spray voltage：3.5kV；Negative ion mode
Spray voltage：2.5kV；Capillary temperature under both of which：250 DEG C, heter temperature：425 DEG C, sheath gas air-flow：50AU, auxiliary
Gas air-flow：13AU, blowback air air-flow：0AU；Lens voltage：60V.Using sweeping pattern, sweep limits entirely：70 arrive 1050m/z；Point
Resolution：70000.
3. material is qualitative
Biomarker it is qualitative using with standard items cortisol and dehydrobenzene compare Chromatographic information (retention time) and
Information in Mass Spectra (accurate molecular weight), and the Chromatographic information of Isotopic Internal Standard standard items series in sample is compared in real time to correct reservation
Time.
4. data analysis：
Biomarker screening confirms key metabolites using Multivariate Logistic Regression.
5. cortisol and dehydrobenzene difference and diagnosis are anticipated in healthy control group, idiopathic male infertility group serum sample
Justice.
The information of corrected age, constitutional index, smoking and history of drinking history, logistic regression finds serum
Cortisol and dehydrobenzene and idiopathic male infertility closely related (Fig. 1).
The stability analysis of cortisol and dehydrobenzene in the serum of embodiment 3
(interval is evaluated the stability of cortisol in serum and dehydroepiandrosterolevels levels using the method for embodiment 2
Time is 2 weeks).As a result show, cortisol and dehydrobenzene determine horizontal stable (Fig. 2) in serum, possess as diagnosis/prison
Survey the characteristic of mark.
The diagnosis of cortisol and dehydrobenzene combination to idiopathic male infertility in the serum of embodiment 4
According to above-mentioned UPLC-Q exactive MS metabolism group methods, the present inventor by the case of random crowd 15 and
The blood serum sample detection cortisol of 15 controls and dehydrobenzene, draw ROC curve and assess sensitivity and the spy of diagnosis with this
The opposite sex, and then assess this diagnosis capability of 2 levels of substance to idiopathic male infertility in detection serum.
The sensitivity of cortisol is 86.67%, and specificity is 86.67%, and area is 0.8933 (Fig. 3) under ROC curve；It is de-
The sensitivity of hydrogen meter androsterone is 73.33%, and specificity is 80.00%, and area is 0.8089 (Fig. 4) under ROC curve.
The sensitivity of combination cortisol and dehydrobenzene is 86.67%, and specificity is 93.33%, area under ROC curve
For 0.9244 (Fig. 5).
So combination cortisol and dehydrobenzene have the ability for preferably diagnosing idiopathic male infertility.
Embodiment 5 is used for cortisol and dehydrobenzene detection and diagnostic kit in idiopathic male infertility serum
Make
Have in determining normal control and idiopathic male infertility serum by the method for UPLC-Q exactive MS first
There is more abundant metabolism small molecule.Then, wherein by the metabonomic technology sieve based on UPLC-Q exactive MS
The choosing biomarker related to idiopathic male infertility, as the diagnosis index for being whether idiopathic male infertility.It is preferred that will
The quantity of the corresponding serum biomarkers for filtering out is controlled at 2, and this is make on the basis of preliminary experiment optimized
Simplify.Using cortisol in serum and dehydrobenzene this 2 biomarkers, both can ensure preferable sensitivity with it is special
Degree, and can be cost-effective, mitigate the burden of patient, moreover it is possible to reduce detection time, have the advantages that quick, accurate, economical, it is easy to
Clinical expansion is used, certainly using wherein 1 mark can also, it is more preferable using 2 mark effects.This kit includes one
Criticize serum cortisol and dehydrobenzene detection reagent and consumptive material, the wherein qualitative and quantitative employing cortisol of biomarker
With dehydrobenzene standard items, assistant analysis are using internal standard A：Creatinine, valine, nicotinic acid, thymidine, glutaric acid, L- phenyl
Alanine, N- acetaminophens, the deuterium mark Isotopic Internal Standard of eight kinds of materials of hippuric acid.Internal standard B：The same position of deuterium mark of pentadecanoic acid
Plain internal standard.Internal standard C：The deuterium mark Isotopic Internal Standard of lignoceric acid.Other are also used for the supporting reverse color of UPLC chromatographic isolations
Spectrum post (Hypersil GOLD C18 chromatographic columns, 100mm × 2.1mm, 1.9 μm of particle diameter), the reagent for precipitating haemocyanin
(100% methyl alcohol), for the reagent (water containing 0.1% formic acid and the acetonitrile containing 0.1% formic acid) of mobile phase, for extracting
The reagent (100% ultra-pure water) of cortisol and dehydrobenzene.The value of this kit is to only need to 10 μ l serum, you can inspection
Survey serum in cortisol and dehydrobenzene mark content, then by content diagnose idiopathic male infertility, and be easy to into
Mobile state is monitored and observation and treatment effect.
Concrete kit forms are as follows：
Cortisol standards
Dehydrobenzene standard items
Internal standard A (creatinine, valine, nicotinic acid, thymidine, glutaric acid, the acid of L- phenylpropylamines, N- acetaminophens, horse
The deuterium mark Isotopic Internal Standard aqueous solution of eight kinds of materials of uric acid)
Internal standard B (the deuterium mark Isotopic Internal Standard methanol solution of pentadecanoic acid)
Internal standard C (the deuterium mark Isotopic Internal Standard methanol solution of lignoceric acid)
Further, can also contain：
Chromatographic column (Thermo 100mm × 2.1mm, 1.9 μm of particle diameter, Hypersil GOLD C18 chromatographic columns)
Reagent A (100% methyl alcohol)
Reagent B (water containing 0.1% formic acid)
Reagent C (acetonitrile containing 0.1% formic acid)
Reagent D (100% ultra-pure water).
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Claims (8)
1. steroid hormone mark related to idiopathic male infertility in serum, it is characterised in that the mark is cortisol
And/or dehydrobenzene.
2. the steroid hormone mark described in claim 1 prepare idiopathic male infertility diagnosis or monitoring reagent in should
With.
3. it is a kind of diagnosis or monitor idiopathic male infertility kit, it is characterised in that the kit contain detection serum in skin
The reagent of matter alcohol and/or dehydrobenzene.
4. kit according to claim 3, it is characterised in that the kit contains using UPLC-Q exactive MS
The reagent of cortisol and/or dehydrobenzene in method detection serum.
5. kit according to claim 4, it is characterised in that the kit contains following reagent：
Cortisol and/or dehydrobenzene standard items；
Internal standard A：Creatinine, valine, nicotinic acid, thymidine, glutaric acid, the acid of L- phenylpropylamines, N- acetaminophens, hippuric acid
In one or more material Isotopic Internal Standard；
Internal standard B：The Isotopic Internal Standard of pentadecanoic acid；
Internal standard C：The Isotopic Internal Standard of lignoceric acid.
6. kit according to claim 5, it is characterised in that the kit also contains：
Hypersil GOLD C18 chromatographic columns；
Reagent A：100% methyl alcohol；
Reagent B：Water containing 0.1% formic acid；
Reagent C：Acetonitrile containing 0.1% formic acid；
Reagent D：Ultra-pure water.
7. steroid hormone mark related to idiopathic male infertility in a kind of detection serum as claimed in claim 1
Method, it is characterised in that the method adopts UPLC-Q exactive MS methods, and cortisol and/or dehydrogenation table are male in detection serum
Ketone content.
8. method according to claim 7, it is characterised in that in the method：
First, liquid-phase condition：
Liquid-phase chromatographic column is Hypersil GOLD C18 chromatographic columns, and column temperature is 40 DEG C；
Mobile phase A is the water containing 0.1% formic acid, and Mobile phase B is the acetonitrile containing 0.1% formic acid, and flow velocity is 400 μ L/min；
Instrument gradient is：0-3min 1%B, 3-10min 1% arrive 99%B, 10-13min 99%B, 13-13.1min 99%
To 1%B, 13.1-17min 1%B；
Input mode：The μ l of volume 5；
2nd, Mass Spectrometry Conditions
It is analyzed using heating electron spray ionisation mode (HESI), positive ion mode spray voltage：3.5kV；Negative ion mode
Spray voltage：2.5kV；Capillary temperature under both of which：250 DEG C, heter temperature：425 DEG C, sheath gas air-flow：50AU, auxiliary
Gas air-flow：13AU, blowback air air-flow：0AU；Lens voltage：60V；Using sweeping pattern, sweep limits entirely：70 arrive 1050m/z；Point
Resolution：70000.
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| CN107064400A (en) * | 2017-04-20 | 2017-08-18 | 博厚健康科技股份有限公司 | The method for detecting five steroids hormones in serum simultaneously |
| CN112129877A (en) * | 2020-09-24 | 2020-12-25 | 南京医科大学 | Seminal plasma mannose-6-phosphate and neopterin detection as idiopathic male sterility diagnostic marker and application thereof |
| CN115554403A (en) * | 2022-08-16 | 2023-01-03 | 山东大学 | Application of steroid hormone DHEA as receptor ADGRG2 agonist ligand |
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| CN107064400A (en) * | 2017-04-20 | 2017-08-18 | 博厚健康科技股份有限公司 | The method for detecting five steroids hormones in serum simultaneously |
| CN112129877A (en) * | 2020-09-24 | 2020-12-25 | 南京医科大学 | Seminal plasma mannose-6-phosphate and neopterin detection as idiopathic male sterility diagnostic marker and application thereof |
| CN112129877B (en) * | 2020-09-24 | 2021-07-20 | 南京医科大学 | Seminal plasma mannose-6-phosphate and neopterin detection as idiopathic male sterility diagnostic marker and application thereof |
| CN115554403A (en) * | 2022-08-16 | 2023-01-03 | 山东大学 | Application of steroid hormone DHEA as receptor ADGRG2 agonist ligand |
| CN115554403B (en) * | 2022-08-16 | 2024-03-08 | 山东大学 | Use of the steroid hormone DHEA as receptor ADGRG2 agonist ligand |
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