CN109946411A - Biomarker and its screening technique for OsificationoftheLigamentumFlavumoftheThoracicSpine diagnosis - Google Patents
Biomarker and its screening technique for OsificationoftheLigamentumFlavumoftheThoracicSpine diagnosis Download PDFInfo
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Abstract
The invention discloses the biomarker diagnosed for OsificationoftheLigamentumFlavumoftheThoracicSpine, the biomarker is lysophosphatidyl choline, and the biomarker lysophosphatidyl choline is C24H50NO7P、C26H52NO6P、C26H54NO7P and C25H52NO7One of P or a variety of.The invention also discloses a kind of screening techniques of biomarker for OsificationoftheLigamentumFlavumoftheThoracicSpine diagnosis.The present invention obtains the biomarker and its screening technique diagnosed for OsificationoftheLigamentumFlavumoftheThoracicSpine using blood serum metabolic omics technology and data statistic analysis technology, strong operability, accuracy is high, by taking blood to can be achieved with diagnosing, rapid and convenient, cost is low, and biomarker of the invention is utilized to diagnose OsificationoftheLigamentumFlavumoftheThoracicSpine specificity with higher, early discovery, the early treatment for being conducive to OsificationoftheLigamentumFlavumoftheThoracicSpine, have good clinical use and promotional value.
Description
Technical field
The present invention relates to field of biological detection, and in particular to the biomarker diagnosed for OsificationoftheLigamentumFlavumoftheThoracicSpine and
Its screening technique.
Background technique
Ossification of ligamenta flava (Ossification of ligamentum flavum, OLF) is a kind of occurs in people's ridge
The ectopic ossification disease of column ligamentum flavum, the lesion can cause the compressing at spinal cord rear, cause quadriplegia, fecal and urinary functions barrier
Hinder, finds that cervical vertebra, thoracic vertebrae and lumbar vertebrae have ossification of the yellow ligament phenomenon from from a large amount of clinical cases.OsificationoftheLigamentumFlavumoftheThoracicSpine
(thoracic ossification of ligamentum flavum, TOLF) is that the ligamentum flavum of thoracic vertebrae intraspinal tube ossify
Lead to spinal canal stenosis, so spinal cord oppressed after a series of clinical symptoms for generating, as lower limb numbness and cacesthesia, under
Myasthenia of limbs even steps on cotton sense and zonesthesia.The pathogenesis of TOLF it is not immediately clear that Most scholars think that it may
It is related with the factors such as chronic injury, regression, inflammation and metabolism, because this disease is easily sent out in the crowd of long campaigns heavy physical labour
It is raw, and this disease takes place mostly in middle Thoracic, this is related greatly with the activity of middle Thoracic, so that ligamentum flavum is in these position institutes
The stress received is larger and easily causes phenomenon of ossify.
Metabolism group (Metabolomics/Metabonomics) is most active in systems biology research field in recent years
One of subdiscipline, be the important composition portion of the another systems biology after genomics, transcription group, proteomics
Point.Organism is a complete system, and body tissue and its regulation are horizontal interrelated and interdepend and outer by environment etc.
The influence of boundary's factor, any variation of vital movement can cause the variation of organism metabolin.Metabolism group passes through analysis
The metabolite of biological fluid and tissue studies the regulation feature of biochemical type system and biology entirety.Its advantage is as follows: gene
And the minor change of albumen can be amplified in metabolite level；Metabolites kinds are far fewer than gene and protein number；Inspection
Survey method takes human body fluid tissue, is hurtless measure.By metabolism group research it can be found that organism is in various internal and external environments
Response situation after variation, can also distinguish the difference between Different Individual of the same race.Metabolism group is using highly sensitive, high-throughput
Modern instrumental analysis means, it is qualitative, quantitatively study organism occurrence and development during metabolin variation, screening and identify
Biomarker has important value to clinical diagnosis and prognostic monitoring, shows wide potential applicability in clinical practice.
Summary of the invention
In order to realize early detection, the early intervention of OsificationoftheLigamentumFlavumoftheThoracicSpine, the purpose of the present invention is to provide a kind of use
In the biomarker of OsificationoftheLigamentumFlavumoftheThoracicSpine diagnosis.
The second object of the present invention is to provide the biomarker in the kit of preparation diagnosis OsificationoftheLigamentumFlavumoftheThoracicSpine
In application.
The third object of the present invention is to provide a kind of screening side of biomarker for OsificationoftheLigamentumFlavumoftheThoracicSpine diagnosis
Method.
To achieve the above object, present invention firstly provides it is a kind of for OsificationoftheLigamentumFlavumoftheThoracicSpine diagnosis biomarker,
The biomarker is lysophosphatidyl choline.
Preferably, the lysophosphatidyl choline is C24H50NO7P、C26H52NO6P、C26H54NO7P and C25H52NO7In P
It is one or more.
Preferably, the biomarker is serum markers.
Document has been delivered in comparison, and metabolin lysophosphatidyl choline is to find in OsificationoftheLigamentumFlavumoftheThoracicSpine for the first time, this is right
It is of great significance in the diagnosing and treating of OsificationoftheLigamentumFlavumoftheThoracicSpine.Lysophosphatidyl choline is internal lecithin lipid metaboli
Lecithin in blood plasma can be become lysolecithin under lecithin cholesterol acyltransferase catalysis by intermediate product.Pass through text
Offer investigation, it has been found that lysophosphatidyl choline plays the role of the Atherogenic factor for facilitating osteoporosis to develop
(Abdallah Fallah,ed.Lysophosphatidylcholine-induced cytotoxicity in
osteoblast-like MG-63cells:Involvement of transient receptor potential
vanilloid 2(TRPV2)channels.Molecular Membrane Biology,2013；30 (5-6): 315-326),
Therefore lysophosphatidyl choline LysoPC (18:0) and bone metabolism have close relationship.
The present invention passes through it has furthermore been found that the biomarker C24H50NO7P、C26H52NO6P、C26H54NO7P、C25H52NO7P
It is reduced in OsificationoftheLigamentumFlavumoftheThoracicSpine patients serum compared to the expression quantity in normal healthy controls person's serum.
Further, the present invention provides the biomarkers in the kit for preparing diagnosis OsificationoftheLigamentumFlavumoftheThoracicSpine
In application.
Preferably, the kit includes detection C24H50NO7P、C26H52NO6P、C26H54NO7P and C25H52NO7One in P
The reagent of kind or a variety of metabolite markers object concentration.
Further, the present invention provides a kind of screening sides of biomarker for OsificationoftheLigamentumFlavumoftheThoracicSpine diagnosis
Method, comprising the following steps:
(1) OsificationoftheLigamentumFlavumoftheThoracicSpine patient and normal healthy controls person's serum sample sample collection: are collected；
(2) liquid chromatography mass acquires: carrying out pre-separation to sample by liquid chromatogram, mass spectrum acquires primary ion and two
Grade daughter ion information；
(3) initial data, correction mass point pattern recognition analysis metabolite profile: are handled using Progenesis QI software
Several and retention time, is grouped sample, imports the progress principal component analysis of EZinfo software and orthogonal Partial Least Squares-is sentenced
It does not analyze, the OPLS-DA model that orthogonal ginsenoside obtains is tested with cross-validation method；
(4) screen: the variable of the OPLS-DA model obtained according to above-mentioned orthogonal ginsenoside is important
Property scoring and P value carry out the screening of difference metabolin, screening criteria are as follows: value<0.05 VIP>2, P；
(5) it identifies: library identification is searched by mankind's metabolism group database and American National Standard and Technical Board chemline
Difference metabolin.
Preferably, the method also includes using Hierarchical Cluster Analysis, metabolic pathway analysis method, ROC curve point
One of analysis method or a variety of methods further screen the difference biomarker screened.
Preferably, the serum sample based on liquid chromatogram and mass spectrographic method acquisition data when, every 10 samples it
Between be inserted into a Quality control samples, for during Real Time Monitoring sample analysis quality control situation.
Preferably, carry out the following processing before the sample sample introduction: the second that 300uL is pre-chilled at -20 DEG C is added in 100uL serum
Nitrile after vortex oscillation 1min, is stood overnight in -20 DEG C；Then in centrifuge at 4 DEG C with 1,2000rpm, be centrifuged 20min,
Take supernatant, the dilution of 1 × water.
Preferably, chromatographic column used in liquid chromatogram is Waters HSS T3column chromatographic column, and column temperature is 45 DEG C, sample
Temperature is 4 DEG C, liquid phase flow rate 0.45ml/min, and chromatogram flow phase includes two kinds of solvent As and B: mobile phase A is 0.1% formic acid water
Solution, Mobile phase B are the acetonitrile solution of 0.1% formic acid, chromatography condition of gradient elution are as follows: 0-1min 1%B, 1-1.5min are
1%B-20%B is gradually incremented by, and 1.5-11min is that 20%B-99%B is gradually incremented by, and 11-11.5min is that 99%B is kept to 98%B,
11.5-14 balancing chromatographic column for 1%B；
Mass Spectrometer Method uses quadrupole rod time-of-flight mass spectrometry instrument Q-TOF, and using the anion of electric spray ion source
Mode ESI-, capillary voltage 2.5kV, orifice potential 25V, ion source temperature are 100 DEG C, remove solvent stream speed 600L/
H, taper hole gas velocity 10L/h, the mass charge ratio range of spectrum data acquisition are 50~2000m/z, and the scan frequency of acquisition is 0.2s/
Circulation.
Further, the answering in diagnosis thoracic vertebrae OsificationoftheLigamentumFlavumoftheThoracicSpine the present invention also provides the screening technique
With.
Beneficial effects of the present invention are as follows:
The present invention is obtained using blood serum metabolic omics technology and data statistic analysis technology for OsificationoftheLigamentumFlavumoftheThoracicSpine
The biomarker and its screening technique of diagnosis, strong operability, accuracy is high, by taking blood to can be achieved with diagnosing, quickly just
Victory, cost is low, utilizes biomarker C of the invention24H50NO7P、C26H52NO6P、C26H54NO7P and C25H52NO7P diagnoses thoracic vertebrae
Ossification of the yellow ligament specificity with higher is conducive to early discovery, the early treatment of OsificationoftheLigamentumFlavumoftheThoracicSpine, has good clinic
Value for applications.
Detailed description of the invention
Fig. 1 principal component analysis shot chart；C, qc, T respectively represent healthy control group sample, quality control sample sheet and TOLF
Case group sample；
Fig. 2 .OPLS-DA shot chart；C (square) indicates that healthy control group sample, T (triangle) indicate TOLF case group sample
This.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, reagent used can be commercially available.
Test method without specific conditions in embodiment, usually conventional method in that art, such as according to normal conditions
Extract serum.
The information of the main agents and instrument mentioned in following embodiment is shown in Tables 1 and 2:
1 main agents of table
| Reagent | CAS | Purity | Brand |
| Water | 7732-18-5 | LC-MS | Thermo |
| Acetonitrile | 75-05-8 | LC-MS | Thermo |
| Formic acid | A117-50 | LC-MS | Thermo |
2 key instrument of table
| Instrument | Model | Brand |
| Ultra high efficiency liquid phase | WatersACQUITYUPLCI-Class | Waters |
| High resolution mass spec | WatersXevoG2-XSQtof | Waters |
| Chromatographic column | WatersHSST3column | Waters |
| Centrifuge | LegendMicro17R | Thermo |
Term used herein TOLF is OsificationoftheLigamentumFlavumoftheThoracicSpine (thoracic ossification of
Ligamentum flavum) english abbreviation.
The present inventor utilizes ultra performance liquid chromatography-level four bars-flight time-tandem mass spectrum (UPLC-Q-TOF-
MS) platform carries out the detection and analysis of metabolin to the serum sample of OsificationoftheLigamentumFlavumoftheThoracicSpine (TOLF) patient and normal healthy controls person,
Using statistical analysis, C is filtered out24H50NO7P、C26H52NO6P、C26H54NO7P and C25H52NO7P is difference metabolin,
C24H50NO7P、C26H52NO6P、C26H54NO7P and C25H52NO7P is related with OsificationoftheLigamentumFlavumoftheThoracicSpine, prompts above-mentioned biomarker
There may be good additive diagnostic value, the diagnostic marker of OsificationoftheLigamentumFlavumoftheThoracicSpine can be become.
Embodiment 1 is based on UPLC-Q-TOF-MS Platform Screening difference biomarker
1, sample collection
Select the specific TOLF patient of diagnosis as TOLF case group, healthy population is being known as healthy control group
Collection after feelings agreement to two groups of carry out blood samples, the sample being collected into are immediately placed in -80 DEG C of low temperature refrigerators and store.TOLF disease
Example group collects 25 parts of blood samples altogether, and healthy control group collects 24 blood samples altogether.3-5 days before acquisition blood sample, to measure crowd
Standard diet is provided.
TOLF case group is included in standard:
(1) it is clarified a diagnosis according to medical history, clinical manifestation, auxiliary examination as TOLF；
(2) have complete clinical data and imaging data and environmental factor assessment；
(3) informed consent is signed；
(4) Chinese han population.
TOLF case group exclusion criteria:
(1) other causes of disease lead to thoracic spinal stenosis person, including scoliosis, humpback, spine fracture etc.；
(2) there are generalized metabolic disease, such as fluorosis of bone, low-phosphorous anti-D malacosteon；
(3) mother has clear medication history, toxicant exposure history, high temperature exposure history person the pregnancy period；
(4) epidemiologic data, clinical data and the imperfect person of image data；
(5) clinical phenotypes meet known clinical congenital syndrome person；
(6) informed consent person is not obtained.
Healthy control group is included in standard:
Gender, age match with TOLF case group, and obtain the healthy population of informed consent.
Healthy control group exclusion criteria:
(1) there is deformity of spine medical history；
(2) other spinal diseases patient, such as spinal trauma, infection, tumour；
(3) there are general metabolism disease, such as fluorosis of bone, low-phosphorous anti-D malacosteon；
(4) mother has clear medication history, toxicant exposure history, high temperature exposure history person the pregnancy period；
(5) clinical phenotypes meet known clinical congenital syndrome person；
(6) informed consent is not obtained.
2, the processing of sample:
(1) sample freezed is placed in thaws at room temperature, takes 100uL serum that 300uL acetonitrile (ACN) (- 20 DEG C of pre-coolings) is added,
After vortex oscillation 1min, stood overnight in -20 DEG C；
(2) then in centrifuge at 4 DEG C with 1,2000rpm, be centrifuged 20min, take supernatant, the dilution of 1 × water takes wherein
100ul is to sample injection bottle；
(3) respectively 50ul is taken to mix, made QC sample (quality control sample sheet).
3, liquid chromatography mass combination analysis
(1) chromatographic condition: 45 DEG C of column temperature, 4 DEG C of sample temperature, liquid phase flow rate 0.45ml/min, mobile phase A: water+0.1%
Formic acid, Mobile phase B :+0.1% formic acid of acetonitrile, gradient elution program are as shown in table 3.
3 gradient elution program of table
| Time (min) | A% | B% | Curve |
| Starting | 99 | 1 | Starting |
| 1 | 99 | 1 | 6 |
| 1.5 | 80 | 20 | 6 |
| 11 | 1 | 99 | 6 |
| 11.5 | 2 | 98 | 6 |
| 14 | 99 | 1 | 1 |
(2) Mass Spectrometry Conditions:
ESI ion source, Masslynx software is based on MS under cation acquisition modeEFunction carries out level-one, second level to sample
Mass spectrometric data acquisition.
Capillary voltage: 2.5kV, orifice potential 24V, remove solvent stream speed 800L/h, taper hole by 100 DEG C of ion source temperature
The ion that m/z is 50-2000Da is scanned in gas velocity 50L/h, 14min, 0.2sec/ circulation.
(3) quality control of the experiment
Process control: when blood sample carries out metabolism group detection, stability is done with QC sample before machine on official sample
The same QC sample is repeated 10 needle of sample introduction or so by test, this sample is condition QC, the sample introduction after instrument stabilizer.Into
When sample, a QC sample is inserted into every 10 this centre of needle-like, and the QC sample being inserted among sample is done peak alignment, determines retention time
It is held essentially constant with peak intensity, instrument stabilizer.
Data control: for the repeatability for investigating data acquisition, correlation is carried out to the metabolin peak intensity of QC sample
Analysis, related coefficient are all larger than 0.90, it is shown that fabulous consistency.In addition, carrying out PCA analysis to all samples, reaction is real
Test the repeatability in otherness and group between group.QC sample is relatively concentrated, and instrument is more stable.
4, data processing
After obtaining original Metabolic fingerprinting, initial data importing Progenesis QI (Waters) software is united
Meter analysis carries out mass fraction correction to data by enkephalins first, peak area corrected retention time is extracted, then to sample
It is grouped, imports the screening that EZinfo software carries out difference metabolin.Using orthogonal ginsenoside
(OPLS-DA) pattern recognition analysis is carried out to healthy control group metabolite profile and OsificationoftheLigamentumFlavumoftheThoracicSpine group metabolite profile, in conjunction with
VIP and P value screens difference metabolin, and does further ROC curve screening and path analysis determination to difference biomarker
Potential biomarker.
5, spectrum analysis and potential biomarker are metabolized
(1) principal component analysis (PCA)
PCA analysis is the first step of EZinfo software analysis, mainly carries out Dimension Reduction Analysis to data, can detecte experiment
The repeatability in otherness and group between group more can intuitively describe the difference between sample.Fig. 1 is pca model, and C, qc, T divide
Three kinds of TOLF case group sample, quality control sample sheet and healthy control group sample sample packets are not represented, and each circle represents this
Be incident upon the position on two-dimensional surface after the metabolism group dimension-reduction treatment of one sample, between the percentage expression group in transverse and longitudinal coordinate
The percentage of analysis result comprehensively, the differentiation of the bigger expression of percentage in that direction can be explained in the difference in this direction
It is better to spend, and shows that the metabolism spectrum of TOLF case group and healthy control group has apparent difference.Repeated preferable experiment, it is same
Different samples in group should be gathered in the range of a Relatively centralized, and can be distinguished with the data aggregation zone of other groups
It opens.
(2) orthogonal ginsenoside (OPLS-DA)
Healthy control group and TOLF case group are distinguished using OPLS-DA method, choose OPLS-DA analysis filtering and classification
Incoherent signal obtains OPLS-DA model, is tested to the quality of model with cross-validation method, Fig. 2 obtains for OPLS-DA
Component, c (square) indicate healthy control group sample, t (triangle) indicate TOLF case group sample, from sample packet as can be seen that
TOLF case group and healthy control group are significantly distinguished on t [1] direction.
VIP (variable importance of OPLS-DA modeling scores) marking can be carried out to metabolin by model analysis to screen,
The higher metabolin of VIP score, it is bigger to the contribution of grouping, choose VIP>2, the metabolin of Pvalue<0.05 is difference metabolism
Object carries out searching library, passes through mankind's metabolism group database (HMDB, http://www.hmdb.ca/) and American National Standard and technology
Office's chemline (NIST, http://webbook.nist.gov/chemistry/) is searched in matching identification blood serum metabolic spectrum
Metabolin, 25 difference metabolins are obtained.
6, difference marker diagnostic evaluation-ROC curve analysis
ROC mapping is carried out to the 25 difference metabolins screened, disease group and reference group measurement result are divided
Analysis, determine the bound of measured value, group away from and point of cut-off (cut-offpoint), list accumulation frequency away from interval by the group of selection
Number distribution table calculates separately out the sensibility, specificity and false positive rate (1- specificity) of all point of cut-offs.It is vertical with sensibility
Coordinate represents true positive rate, and (1- specificity) is that abscissa represents false positive rate, and mapping is drawn in ROC curve, and (subject works bent
Line).Area value (AUC) under ROC curve is between 0.5-1, closer to 1, illustrates that diagnosis effect is better.AUC 0.5~
There is lower accuracy when 0.7, AUC has certain accuracy at 0.7~0.9, and AUC has high accuracy at 0.9 or more.AUC
When=0.5, illustrate that diagnostic method does not work completely, no diagnostic value.
7, result
By analyzing above, it has been found that metabolin lysophosphatidyl choline C24H50NO7P、C26H52NO6P、C26H54NO7P
And C25H52NO7P has notable difference in TOLF case group and healthy control group.The specifying information of 4 difference metabolins such as table 4
It is shown:
44 difference metabolin information of table
C24H50NO7P、C26H52NO6P、C26H54NO7P and C25H52NO7The VIP value of P is all larger than 2, P value and is respectively less than 0.05,
C24H50NO7P、C26H52NO6P、C26H54NO7P and C25H52NO7P has notable difference in TOLF case group and healthy control group,
And healthy control group is substantially less than in the expression quantity of TOLF case group.
C24H50NO7P、C26H52NO6P、C26H54NO7P and C25H52NO7The AUC value of PROC curve is all larger than 0.7.It is possible thereby to
Find out, the serum protein moteblites lysophosphatidyl choline C that the present invention screens24H50NO7P、C26H52NO6P、C26H54NO7P and
C25H52NO7The diagnosis effect of P is more significantly, with C24H50NO7P、C26H52NO6P、C26H54NO7P and C25H52NO7One in P
Kind or a variety of diagnose as marker progress OsificationoftheLigamentumFlavumoftheThoracicSpine have certain value of clinical studies.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. the biomarker for OsificationoftheLigamentumFlavumoftheThoracicSpine diagnosis, which is characterized in that the biomarker is lysophosphatide
Phatidylcholine.
2. biomarker as described in claim 1, which is characterized in that the lysophosphatidyl choline is C24H50NO7P、
C26H52NO6P、C26H54NO7P and C25H52NO7One of P or a variety of.
3. biomarker as described in claim 1, which is characterized in that the biomarker is serum protein moteblites.
4. biomarker as claimed in any one of claims 1-3 is in the kit of preparation diagnosis OsificationoftheLigamentumFlavumoftheThoracicSpine
Application.
5. application as claimed in claim 4, it is characterised in that the kit includes detection C24H50NO7P、C26H52NO6P、
C26H54NO7P and C25H52NO7The reagent of one of P or a variety of biomarker concentrations.
6. a kind of screening of the biomarker as claimed in any one of claims 1-3 for OsificationoftheLigamentumFlavumoftheThoracicSpine diagnosis
Method, which comprises the following steps:
(1) OsificationoftheLigamentumFlavumoftheThoracicSpine patient and normal healthy controls person's serum sample sample collection: are collected；
(2) liquid chromatography mass acquires: carrying out pre-separation to sample by liquid chromatogram, mass spectrum acquires level-one parent ion and second level
Daughter ion information；
(3) pattern recognition analysis metabolite profile: using Progenesis QI software handle initial data, correction mass score and
Retention time is grouped sample, imports EZinfo software and carries out principal component analysis and orthogonal Partial Least Squares-differentiation point
Analysis, the OPLS-DA model that orthogonal ginsenoside obtains are tested with cross-validation method；
(4) screen: the variable importance of the OPLS-DA model obtained according to above-mentioned orthogonal ginsenoside is commented
Divide and P value carries out the screening of difference metabolin, screening criteria are as follows: value<0.05 VIP>2, P；
(5) it identifies: library being searched by mankind's metabolism group database and American National Standard and Technical Board chemline and identifies difference
Metabolin.
7. screening technique as claimed in claim 6, which is characterized in that the method also includes using Hierarchical clustering analysis side
One of method, metabolic pathway analysis method, ROC curve analysis method or a variety of methods are to the difference metabolin screened into one
Step screening.
8. screening technique as claimed in claim 6, which is characterized in that when the liquid chromatography mass acquires, every 10 samples
A Quality control samples are added, for Real Time Monitoring sample from sample introduction pre-treatment to analytic process in quality control feelings
Condition.
9. screening technique as claimed in claim 6, which is characterized in that carried out the following processing before the sample sample introduction: 100uL blood
Reset and add the acetonitrile being pre-chilled into 300uL at -20 DEG C, after vortex oscillation 1min, is stood overnight in -20 DEG C；1 in 4 DEG C of centrifuges,
2000rpm is centrifuged 20min, takes supernatant, the dilution of 1 × water.
10. screening technique as claimed in claim 6, which is characterized in that chromatographic column used in liquid chromatogram is Waters HSS T3
Column chromatographic column, column temperature are 45 DEG C, and sample temperature is 4 DEG C, liquid phase flow rate 0.45ml/min, and chromatogram flow phase includes two kinds molten
Agent A and B: mobile phase A is 0.1% aqueous formic acid, and Mobile phase B is the acetonitrile solution of 0.1% formic acid, chromatography condition of gradient elution
Are as follows: 0-1min 1%B, 1-1.5min gradually increase for 1%B-20%B, and 1.5-11min is that 20%B-99%B is gradually incremented by,
11-11.5min is that 98%B is kept to by 99%B, and 11.5-14min is that 1%B balances chromatographic column；
Mass Spectrometer Method uses quadrupole rod time-of-flight mass spectrometry instrument Q-TOF, and using the negative ion mode of electric spray ion source
ESI-, capillary voltage 2.5kV, orifice potential 24V, ion source temperature are 100 DEG C, remove solvent stream speed 800L/h, bore
The mass charge ratio range of gas flow hole speed 50L/h, spectrum data acquisition are 50~2000m/z, and the scan frequency of acquisition is followed for 0.2s/
Ring.
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| CN111208229A (en) * | 2020-01-17 | 2020-05-29 | 华中农业大学 | Screening method of serum metabolic marker for low bone density joint diagnosis of laying hens and application of serum metabolic marker |
| CN111413431A (en) * | 2020-04-17 | 2020-07-14 | 中国药科大学 | Method for screening metabolic markers of drug-induced acute kidney injury lesion process |
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