CN107576747A - Capric acid and prostaglandin E2 combination are used as macrosomia's auxiliary diagnosis mark and its application - Google Patents

Abstract

The invention belongs to analytical chemistry and clinical medicine domain, discloses capric acid and prostaglandin E2 combination is used as macrosomia's auxiliary diagnosis mark and its application.The auxiliary diagnosis mark is the combination of metabolism small molecule capric acid and prostaglandin E2, available for macrosomia's auxiliary diagnosis or monitoring reagent box is prepared, has preferable sensitivity and specificity, detection is quick, has higher clinical value.

Description

Capric acid and prostaglandin E2 combination are used as macrosomia's auxiliary diagnosis mark and its application

Invention field

The invention belongs to analytical chemistry and clinical medicine domain, is related to capric acid and prostaglandin E2 combination is auxiliary as macrosomia Help diagnosis marker and its application.

Background technology

Macrosomia (Macrosomia) refers to that body weight is more than or equal to 4kg neonate in 1 hour after being born.The hair of macrosomia Life all has harm to mother and baby.One of an important factor for macrosomia is difficult labour, it can also make the risk increase for suffering from heart malformations.Now Research finds, macrosomia grow up future trouble obesity probability it is larger, people will be susceptible to suffer from as a variety of diseases such as diabetes, hypertension Group.

The early diagnosis of macrosomia at present is generally basede on some susceptible medical features of mother such as with diabetes, obesity And prolonged pregnancy.But for not possessing the crowd of features described above, the early diagnosis of macrosomia needs to rely on B ultrasound detection.But B Super detection time is longer, checks that price is higher, and radiation of some pregnant woman to ultrasound diagnosis is also more worried.These are all to huge Youngster's diagnosis brings difficulty and serious financial burden is brought to family, thus needs the side for finding new macrosomia's diagnosis badly Method.

Metabolism group (Metabolomics/Metabonomics) is that one to grow up phase late 1990s is new Set up schools section, it be by investigate biosystem hereditary change it is stimulated or disturbance after, the change of its metabolite or its with The change of time, to study a science of biosystem.So-called metabolism group (Metabolome) is the downstream product of genome And final product, it is that some participate in organism metabolism, maintain organism normal function and the small molecule grown The set of compound, mainly relative molecular mass are less than 1000 endogenous small molecule, and these endogenous metabolism small molecules are related to Glycometabolism, energetic supersession, lipid metaboli, amino acid metabolism, nucleic acid metabolism, coenzyme metabolism etc..

Organism under normal condition is a complete system, and the metabolin in biological fluid, cell and tissue is in One stable poised state.There occurs pathological change, this balance to be just broken due to heredity or the reason day after tomorrow for body, generation Thank product and metabolic process and also generate corresponding change.Metabolism small molecule is understood in lysis by metabonomic analysis Change, can help people find relevant biomarker (biomarker) can with the diagnosis and prediction of aided disease, The metabolic pathway that people can be helped to be related in itself by small-molecule substance understands the pathogenesis of disease and carried for medicament research and development For specific target.In recent years, metabolism group achieves in the research of mankind's various diseases in the early diagnosis of disease Many achievements in research being significant, such as angiocardiopathy, diabetes and cancer, correlative theses are published in academic journal 《Nature》、《Nature medicine》、《Journal of hepatology》With《Cancer research》On, show Metabolism small molecule potentiality and value huge in diagnosing human disease.

The common technology of metabolism group research at present includes liquid chromatograph mass spectrography (LC-MS), chromatography of gases-mass spectrum connection With (GC-MS) and nuclear magnetic resonance technique (NMR).Nuclear magnetic resonance technique feature is without destruction, Sample pretreatment letter to component to be measured It is single, but sensitivity is relatively low；Gas chromatography-mass spectrography has preferable sensitivity and reappearance, but typically to use derivatization Method carries out pre-treatment to sample so that experimental procedure becomes complicated.And LC-MS has that sample process is simple, high sensitivity, face The characteristics of bed is practical.UPLC-Q exactive MS are the combinations of high resolution mass spectrum of new generation and ultra high efficiency liquid phase, are had Sensitivity, specificity and the stability stronger compared to traditional LC-MS.So it is metabolized using UPLC-Q exactive MSS The metabonomic analysis of small molecule, if the stable special plasma metabolism small molecule related to macrosomia's morbidity can be found as life Thing mark, and the UPLC-Q exactive MS detection methods of the metabolism small molecule mark of corresponding disease are researched and developed, not only at this Field is in first place in the world, can also create the economic benefit to attract people's attention, also will be to improving China's baby's health level Once strong promotion.

The content of the invention

It is an object of the invention to provide capric acid and prostaglandin E2 combination to be used as macrosomia's auxiliary diagnosis mark.

Another object of the present invention is to provide above-mentioned mark in macrosomia's auxiliary diagnosis or monitoring reagent box is prepared Using.

A further object of the present invention is to provide chromatographic mass spectrometry detection or the diagnostic kit of above-mentioned mark.

The purpose of the present invention is realized by following technical measures：

A kind of macrosomia's auxiliary diagnosis mark, it is characterised in that the mark is metabolism small molecule capric acid and prostaglandin E2 combination.

Application of the described auxiliary diagnosis mark in macrosomia's auxiliary diagnosis or monitoring reagent box is prepared.

A kind of kit for being used for macrosomia's auxiliary diagnosis or monitoring, the kit contain detection capric acid and prostaglandin E2 reagent；Especially the kit contains detects capric acid and prostaglandin E2 using UPLC-Q exactive MS methods Reagent.

Described kit, the wherein kit contain：

Capric acid standard items,

Prostaglandin E2 standard items,

The capric acid that carbon 13 marks,

The prostaglandin E2 that carbon 13 marks.

Further, the kit also contains：

Hypersil GOLD C18 chromatographic columns；

Reagent A：Containing 100% methanol (protein precipitation use)；

Reagent B：Water (mobile phase A) containing 0.1% formic acid；

Reagent C：Acetonitrile (Mobile phase B) containing 0.1% formic acid；

Reagent D：100% ultra-pure water (redissolves and used).

A kind of method for detecting above-mentioned mark, this method is using UPLC-Q exactive MS methods detection capric acid with before Row parathyrine E2.In this method：

First, liquid-phase condition：

Liquid-phase chromatographic column is Hypersil GOLD C18 chromatographic columns (100mm × 2.1mm, 1.9 μm of particle diameter, Thermo Scientific, Germany), column temperature is 40 DEG C；

Mobile phase A is that water (reagent B) and Mobile phase B containing 0.1% formic acid are the acetonitrile (reagent C) containing 0.1% formic acid, stream 400 μ L/min of speed；

Instrument gradient is：0-3min 1%B, 3-10min 1% arrive 99%B, 10-13min 99%B, 13-15min 1% B；(B refers to Mobile phase B, the amount of the amount of mobile phase A and corresponding Mobile phase B totally 100% in each gradient, similarly hereinafter)；

Sample introduction：10μL.

2nd, Mass Spectrometry Conditions：

Heating electron spray ionisation mode (HESI) is analyzed；

Positive ion mode spray voltage：Holotype, injection electric 3.5kV, negative mode, injection electric 2.5kV；For Both of which, capillary temperature are 300 DEG C, and heter temperature is 425 DEG C, and sheath throughput is 50AU, secondary air amount 13AU, Blowback throughput is 0AU, and full scan analysis (70 to 1,050m/z), resolution ratio is arranged to 70,000.

The present invention is described in detail as follows：

The present inventor gathers standard compliant pregnancy period blood sample with S.O.P. (SOP), and systematic collection is complete Crowd's Back ground Information and clinical data, and employ the metabolism group method based on UPLC-Q exactive MS and analyzed.

The experimental method specifically studied mainly includes following components：

First, research object selection and packet foundation

First stage screening stage

Include pregnant and lying-in women totally 97 people at random.

1. the age is between 23 to 36 years old；

2. pregnant week is less than or equal to 41 weeks；

3. case containing macrosomia (birth weight >=4kg, 16 people).

Second stage Qualify Phase

Include case and control pregnant and lying-in women totally 30 people.

A groups：Healthy control group (15 people, 2.5kg≤birth weight<4kg)：

1. the age is between 24 to 36 years old；

2. pregnant week is less than or equal to 41 weeks；

3. without whole body major disease.

B groups：Macrosomia's group (15 people, birth weight >=4kg)：

1. the age is between 24 to 35 years old；

2. pregnant week is less than or equal to 41 weeks；

3. without whole body major disease.

2nd, UPLC-Q exactive MS metabonomic analysis and macrosomia are diagnosed with the screening of metabolism small molecule and checking

1. Sample pretreatment

1. fresh pregnancy period blood centrifuges 5min in centrifuge 3000rpm, the μ L of supernatant 100 are taken to dispense into clean 1.5ml pipes.

2.50 μ L of supernatant add the μ L of water 50 to add 300 μ L methanol (reagent A) protein precipitations again.

3. supernatant is drawn, with nitrogen drying again with vacuum drying.

4. the μ L of dried object 20 redissolve (reagent D).

2. instrument detects

2.1. analytical instrument：UPLC Ultimate 3000system (Dionex) high performance liquid chromatograph；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 mobile phase A is that water (reagent B) and Mobile phase B containing 0.1% formic acid are the acetonitrile (reagent containing 0.1% formic acid C), the μ L/min of flow velocity 400.

2.2.3 instrument gradient is：0-3min 1%B, 3-10min 1% arrive 99%B, 10-13min 99%B, 13- 15min 1%B.Totally 100%) (B refers to Mobile phase B, and the amount of the amount of mobile phase A and corresponding Mobile phase B in each gradient

2.2.4 sample introduction：10μL.

2.3. Mass Spectrometry Conditions：

2.3.1 electron spray ionisation mode (HESI) is heated to be analyzed；

2.3.2 positive ion mode spray voltage：Holotype, injection electric 3.5kV, negative mode, injection electric are 2.5kV；For both of which, capillary temperature is 300 DEG C, and heter temperature is 425 DEG C, and sheath throughput is 50 AU, aids in gas Flow is 13AU, and blowback throughput is 0AU, and full scan analysis (70 to 1,050m/z), resolution ratio is arranged to 70,000.

3. material is qualitative

Metabolism small molecule is qualitative to compare Chromatographic information (retention time) and matter using with standard items capric acid and prostaglandin E2 Spectrum information (accurate molecular weight), and (capric acid, the carbon 13 of the mark of carbon 13 mark Isotopic Internal Standard standard items in comparison sample in real time Prostaglandin E2 stable isotope internal standard compound) Chromatographic information (retention time).

4. data analysis：

Biomarker screening is combined using the sane metabolin relevant with birth weight of multiple linear regression screening, then Key metabolites combination is confirmed using Multivariate Logistic Regression.

5. healthy control group, macrosomia organize the difference and diagnostic significance that small molecule is metabolized in blood sample.

The age of corrected mother, pregnant latter stage BMI, the sex of pregnant week and natus, multiple linear regression analysis hair Capric acid and the birth weight of prostaglandin E2 combination and filial generation are proportionate in existing pregnancy period blood.The age of corrected mother, Pregnant latter stage BMI, pregnant week and natus sex, logistic regression find pregnancy period blood capric acid and prostaglandin The ratio increase of capric acid content and prostaglandin E2 content can significantly improve the risk that filial generation is macrosomia in E2 combinations.Using only Vertical crowd applies above-mentioned metabolism small molecule combinatorial auxiliary diagnosis macrosomia, sensitivity 80.00%, specificity 80.00%, Area is 0.8978 under ROC curve, has higher clinical value.

3rd, diagnostic reagent box preparation method

According to above-mentioned a series of experiments result, the present inventor, which is also prepared for one kind, can be used for macrosomia's dynamic monitoring or auxiliary The kit of diagnosis, the kit includes to be stabilized and detectable capric acid and prostate in measure subject's pregnancy period blood Plain E2 stable isotopes internal standard and capric acid and the standard items of prostaglandin E2.Diagnostic kit also includes small point of a set of plasma metabolism Son extraction and used in chromatograph reagent and equipment.

Beneficial effects of the present invention：

The present inventor compares in normal control and mother's macrosomia pregnancy period blood by using UPLC-Q exactive MS Metabolism small molecule, it was found that exist in pregnancy period blood and can be used for assessing whether mother pregnant macrosomia, there is diagnostic value Plasma metabolism small molecule mark combines, and the UPLC-Q exactive MS of the plasma metabolism small molecule marker detection Using, develop the macrosomia of clinical practice can be easy to diagnose, monitoring reagent box.

The present invention is advantageous in that using pregnancy period plasma metabolism small molecule as the mark that macrosomia evaluates：

(1) plasma metabolism small molecule is a kind of new biomarkers, and it is associated by force with disease outcome, not only stablizes, be micro- Create, be easy to detect, and it is quantitative accurate, the Sensitivity and Specificity of macrosomia's auxiliary diagnosis will be greatly improved, micromolecular life Preventing and treating for bad birth outcomes is started brand-new situation by the successful exploitation of thing mark, for grinding for other diseases biomarker System is offered reference.

(2) plasma metabolism small molecule mark provided by the invention can be used for the auxiliary diagnosis mark of macrosomia, pass through Invasive manner is with regard to energy early stage auxiliary diagnosis macrosomia, so as to provide foundation for the further testing in depth testing of clinician, for quick standard Morbid state and coincident with severity degree of condition, the control prece offer support for taking more personalized in time of patient is really provided, delayed With prevention progression of disease.

(3) present invention is verified using mother's pregnancy period blood sample of macrosomia and normal healthy controls crowd, it was demonstrated that pregnant Capric acid and prostaglandin E2 combination have higher sensitivity and specificity in early stage auxiliary diagnosis macrosomia in phase blood, can make Used for mark.

(4) present invention uses tight, multistage checking and appraisement system, screens a variety of blood generations by preliminary experiment initial stage Thank to small molecule, independent crowd's checking carried out using UPLC-Q exactive MS, ensure that the plasma metabolism biomarker and The reliability of diagnostic method.

(5) UPLC-Q exactive MS technology sample process is simple, and Instrumental Analysis is accurate rapidly, has higher clinic Application value.

Brief description of the drawings

The case of Fig. 1 first stage crowds and the birth weight of control.

The top and bottom of box figure represent the 75th and the 25th hundredths, the upper end to lower end of box figure respectively Representative is up to minimum value, and (-) is median, and (+) is average.

The case of Fig. 2 second stage crowds and the birth weight of control, caption is with reference to figure 1.

Fig. 3 screening stages, the age of corrected mother, pregnant latter stage BMI, the sex of pregnant week and natus, polynary line Property return and logistic regression it is consistent discovery pregnancy period blood capric acid and prostaglandin E2 combination can effectively sentence Disconnected filial generation is the risk of macrosomia.

Fig. 4 metabolism small molecule combinatorial detection level fluctuations (mean ± standard deviation).

Fig. 5 Qualify Phases, the Normal group made using the information of pregnancy period plasma metabolism small molecule combinatorial and macrosomia ROC curve between group.

Embodiment

The invention will be further elaborated by the following examples.

The research object of embodiment 1 selects and packet foundation

The present inventor is from the satisfactory macrosomia's infant of the attached Nanjing healthcare hospital for women ＆ children collection of Nanjing Medical University and just Normal mother's children pregnancy period blood sample (macrosomia and control birth weight difference are shown in Fig. 1, Fig. 2).Neonate after birth is weighed, Body weight is equal to or more than 4kg person in 1 hour after being such as born, that is, is diagnosed as " macrosomia ".First stage incorporates conform at random The 97 pregnant and lying-in women's samples asked, wherein 15 are macrosomia；Second stage includes 30 pregnant and lying-in women's samples, wherein 15 controls 15 Example macrosomia, the screening experiment object as macrosomia's pregnancy period plasma metabolism small molecule biomarker.Specific sample group Standard is as follows：

First stage screening stage

Include pregnant and lying-in women totally 97 people at random.

4. the age is between 23 to 36 years old；

5. pregnant week is less than or equal to 41 weeks；

6. case containing macrosomia (birth weight >=4kg, 16 people).

Second stage Qualify Phase

Include case and control pregnant and lying-in women totally 30 people.

A groups：Healthy control group (15 people, 2.5kg≤birth weight<4kg)：

4. the age is between 24 to 36 years old；

5. pregnant week is less than or equal to 41 weeks；

6. without whole body major disease.

B groups：Macrosomia's group (15 people, birth weight >=4kg)：

4. the age is between 24 to 35 years old；

5. pregnant week is less than or equal to 41 weeks；

6. without whole body major disease.

Embodiment 2：UPLC-MS metabolism group macrosomias biomarker screens

1. Sample pretreatment

1.1. fresh pregnancy period blood centrifuges 5min in centrifuge 3000rpm, takes the μ L of supernatant 100 to dispense to clean 1.5ml EP Guan Zhong.

1.2. 50 μ L of supernatant add the μ L of water 50 to add 300 μ L methanol (reagent A) protein precipitations again.

1.3. supernatant is drawn, with nitrogen drying again with vacuum drying.

1.4. the μ L of dried object 20 redissolve (reagent D).

2. instrument detects

2.1. analytical instrument：UPLC Ultimate 3000 system (Dionex) high performance liquid chromatograph；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 mobile phase A is that water (reagent B) and Mobile phase B containing 0.1% formic acid are the acetonitrile (reagent containing 0.1% formic acid C), the μ L/min of flow velocity 400.

2.2.3 instrument gradient is：0-3min 1%B, 3-10min 1% arrive 99%B, 10-13min 99%B, 13- 15min 1%B.Totally 100%) (B refers to Mobile phase B, and the amount of the amount of mobile phase A and corresponding Mobile phase B in each gradient

2.2.4 sample introduction：10μL.

2.3. Mass Spectrometry Conditions：

2.3.1 electron spray ionisation mode (HESI) is heated to be analyzed；

2.3.2 positive ion mode spray voltage：Holotype, injection electric 3.5kV, negative mode, injection electric are 2.5kV；For both of which, capillary temperature is 300 DEG C, and heter temperature is 425 DEG C, and sheath throughput is 50 AU, aids in gas Flow is 13AU, and blowback throughput is 0AU, and full scan analysis (70 to 1,050m/z), resolution ratio is arranged to 70,000.

3. material is qualitative

Metabolism small molecule is qualitative to compare Chromatographic information (retention time) and matter using with standard items capric acid and prostaglandin E2 Spectrum information (accurate molecular weight), and stable isotope internal standard standard items (mark by capric acid, the carbon 13 of the mark of carbon 13 in comparison sample in real time The prostaglandin E2 of note) Chromatographic information (retention time).

4. data analysis：

Biomarker screening is combined using the sane metabolin relevant with birth weight of multiple linear regression screening, then Key metabolites combination is confirmed using Multivariate Logistic Regression.

5. healthy control group, macrosomia organize the difference and diagnostic significance that small molecule is metabolized in blood sample.

The age of corrected mother, pregnant latter stage BMI, the sex of pregnant week and natus, multiple linear regression analysis hair Capric acid and the birth weight of prostaglandin E2 combination and filial generation are proportionate in existing pregnancy period blood.The age of corrected mother, Pregnant latter stage BMI, pregnant week and natus sex, logistic regression find pregnancy period blood capric acid and prostaglandin The ratio increase of capric acid content and prostaglandin E2 content can significantly improve the risk (figure that filial generation is macrosomia in E2 combinations 3)。

The stability analysis of the plasma metabolism small molecule of embodiment 3

The stability of mother's pregnancy period blood capric acid and prostaglandin E2 combined horizontal is commented using the method for embodiment 2 Valency (interval time is 2 weeks).As a result show, capric acid and prostaglandin E2 combine measured horizontal stable (Fig. 4), possess in blood Characteristic as diagnosis/monitoring mark.

Embodiment 4 is metabolized diagnosis of the small molecule combinatorial to macrosomia

According to above-mentioned UPLC-Q exactive MS metabolism group methods, the present inventor by the independent case of crowd 15 and The pregnancy period blood samples detection capric acid and prostaglandin E2 of 15 controls, ROC curve is drawn with this come understand the combination be used for early stage The sensitivity and specificity of auxiliary diagnosis macrosomia.

Fig. 5 shows that the sensitivity for combining capric acid and prostaglandin E2 is 80.00%, specificity 80.00%, and ROC is bent Area is 0.8978 under line, has higher diagnostic value.

So combining capric acid and prostaglandin E2 has the ability for preferably diagnosing macrosomia.

Embodiment 5 is used for the detection of macrosomia's plasma metabolism small molecule and the making of diagnostic kit

Determine have in normal and macrosomia's infant mother's pregnancy period blood by UPLC-Q exactive MS method first There is the metabolism small molecule compared with high abundance.Then, sieved wherein by the metabonomic technology based on UPLC-Q exactive MS The choosing metabolism small molecule related to macrosomia, as early stage auxiliary diagnosis whether be macrosomia and diagnostic routine index, most The quantity of the corresponding plasma metabolism small molecule filtered out is controlled at 2 afterwards, this be made on the basis of preliminary experiment it is optimal That changes simplifies.This kit includes a collection of pregnancy period plasma metabolism small molecule detection reagent and consumptive material, wherein metabolism small molecule It is qualitative and quantitatively using the standard items of capric acid and prostaglandin E2, assisted quantitative and aid in the qualitative capric acid marked using carbon 13, The stable isotope internal standard compound for the prostaglandin E2 that carbon 13 marks.Other supporting reverse chromatograms also having for UPLC chromatographic isolations Post (Hypersil GOLD C18 chromatographic columns, 100 mm × 2.1mm, 1.9 μm of particle diameter), the reagent (100% for protein precipitation Methanol), for the reagent (water containing 0.1% formic acid and the acetonitrile containing 0.1% formic acid) of mobile phase, it is metabolized for extracting The reagent (100% ultra-pure water) of small molecule.The value of this kit is only to need a small amount of pregnancy period blood, you can detection metabolism is small The content of molecular marker, then by content ratio come the generation of early stage auxiliary diagnosis macrosomia, and be easy to carry out dynamic monitoring With observation therapeutic effect.

Specific kit forms are as follows：

Capric acid standard items

Prostaglandin E2 standard items

The capric acid that carbon 13 marks

The prostaglandin E2 that carbon 13 marks

Further, can also contain：

Chromatographic column (Thermo 100mm × 2.1mm, 1.9 μm of particle diameter, Hypersil GOLD C18 chromatographic columns)

Reagent A (contains 100% methanol)

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. a kind of macrosomia's auxiliary diagnosis mark, it is characterised in that the mark is metabolism small molecule capric acid and prostaglandin E2 Combination.

2. application of the auxiliary diagnosis mark in macrosomia's auxiliary diagnosis or monitoring reagent box is prepared described in claim 1.

3. a kind of kit for being used for macrosomia's auxiliary diagnosis or monitoring, it is characterised in that the kit contains detection capric acid with before Row parathyrine E2 reagent.

4. kit according to claim 3, it is characterised in that the kit contains using UPLC-Q exactive MS Method detects capric acid and the reagent of prostaglandin E2.

5. kit according to claim 4, it is characterised in that the kit contains：

Capric acid standard items,

Prostaglandin E2 standard items,

The capric acid that carbon 13 marks,

The prostaglandin E2 that carbon 13 marks.

6. diagnostic kit according to claim 5, it is characterised in that the kit also contains：

Hypersil GOLD C18 chromatographic columns,

Reagent A：Containing 100% methanol,

Reagent B：Water containing 0.1% formic acid,

Reagent C：Acetonitrile containing 0.1% formic acid,

Reagent D：100% ultra-pure water.

A kind of 7. method for detecting mark as claimed in claim 1, it is characterised in that this method uses UPLC-Q exactive MS methods detect capric acid and prostaglandin E2.

8. according to the method for claim 7, it is characterised in that in this method：

First, liquid-phase condition：

Liquid-phase chromatographic column is Hypersil GOLD C18 chromatographic columns (100mm × 2.1mm, 1.9 μm of particle diameter, Thermo Scientific, Germany), column temperature is 40 DEG C；

Mobile phase A is that water (reagent B) and Mobile phase B containing 0.1% formic acid are the acetonitrile (reagent C) containing 0.1% formic acid, flow velocity 400μL/min；

Instrument gradient is：0-3min 1%B, 3-10min 1% arrive 99%B, 10-13min 99%B, 13-15min 1%B；(B Totally 100%) Mobile phase B is referred to, the amount of the amount of mobile phase A and corresponding Mobile phase B in each gradient；

Sample introduction：10μL；

2nd, Mass Spectrometry Conditions：

Heating electron spray ionisation mode (HESI) is analyzed；

Positive ion mode spray voltage：Holotype, injection electric 3.5kV, negative mode, injection electric 2.5kV；For two kinds Pattern, capillary temperature are 300 DEG C, and heter temperature is 425 DEG C, and sheath throughput is 50AU, secondary air amount 13AU, blowback Throughput is 0AU, and full scan analysis (70 to 1,050m/z), resolution ratio is arranged to 70,000.