CN106370765A - Reversed-phase chromatography time-of-flight mass spectrometry-based laryngocarcinoma urine differential metabolite determining and screening method - Google Patents
Reversed-phase chromatography time-of-flight mass spectrometry-based laryngocarcinoma urine differential metabolite determining and screening method Download PDFInfo
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- CN106370765A CN106370765A CN201610706956.2A CN201610706956A CN106370765A CN 106370765 A CN106370765 A CN 106370765A CN 201610706956 A CN201610706956 A CN 201610706956A CN 106370765 A CN106370765 A CN 106370765A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/89—Inverse chromatography
Abstract
A reversed-phase chromatography time-of-flight mass spectrometry-based laryngocarcinoma urine differential metabolite determining and screening method comprises the following steps: centrifuging urine samples, diluting the centrifuged urine samples, directly introducing every diluted urine sample to a reversed-phase chromatography time-of-flight mass spectrometer, detecting the introduced urine samples, carrying out peak alignment, correction and standardization, and carrying out multivariable statistic analysis and spectrum database retrieval to find out differential metabolites in laryngocarcinoma volunteer's urine and healthy volunteer's urine. The method is a brand new method for identifying the differential metabolites in urine, can reflect the change of the metabolites of laryngocarcinoma patient's urine through the differential metabolites, and is helpful for realizing diagnosis, clinic treatment, prognosis judgment and the pathogenesis research of laryngocarcinoma.
Description
Technical field:
The invention belongs to the physical and chemical inspection technical field of urine, specifically a kind of larynx based on reversed phase chromatography flight time mass spectrum
The mensure screening technique of cancer urine difference metabolite.
Background technology:
Laryngeal carcinoma is one of modal malignant tumor of incidence, and sickness rate accounts for the 5% of whole body malignant tumor, in recent years sickness rate
In rising trend, and male's sickness rate is higher than women, and laryngeal carcinoma age of onset, how more than 40 years old, was wherein fallen ill with 60 ~ 70 years old
Rate is highest, and the sickness rate in China's North, Northeast China area is far above each province on the south the Changjiang river.At present, predict the gold of laryngeal pathological process
Standard is that laryngoscope adds histopathology evaluation, but the method has traumatic and spends higher.Metabolism group is the gene that continues
The subject that after group and proteomics, newly-developed gets up, is in specific physiological periods to a certain biological or cell
Interior all low-molecular-weight metabolites carry out new disciplines of qualitative and quantitative analysis simultaneously.Metabonomic technology can be found that
New biomarker, the research of diagnosis, clinical treatment, Index for diagnosis and its pathogenesis that this will be helpful to head and neck cancer.
Urine is a kind of biofluid being widely used, studying, and it has wide material sources, collects convenient, noinvasive, and body
Long-pending than larger the advantages of, be a kind of preferably to detect medium.Because in urine, major part is some nonpolar and middle polarity
Compound, such as lipid, aminoacid etc., reversed phase chromatography has to these compounds and is effectively maintained and separating power, especially with
Combination-the flight time mass spectrum of time of-flight mass spectrometer has high sensitivity, resolution and mass accuracy, can reduce number
According to complexity so that the qualitative ability of flight time mass spectrum is further lifted.At present, using reversed-phase liquid chromatography
The document that flight time mass spectrum is studied to laryngeal carcinoma urine metabolism group have not been reported.
Content of the invention:
The purpose of the present invention is intended to overcome prior art defect, sets up a kind of screening laryngeal carcinoma simple, quick, that selectivity is good and volunteers
The liquid chromatograph flight time mass spectrum method of difference metabolite in person and healthy volunteer's urine, the method can quick, accurate be reflected
Determine the difference metabolite in urine.
The purpose of the present invention is achieved through the following technical solutions:
A kind of mensure screening technique of the laryngeal carcinoma urine difference metabolite based on reversed phase chromatography flight time mass spectrum, is with laryngeal carcinoma will
Hope person and healthy volunteer's urine are measured for sample, compare, screen, and specifically include following processing step:
A, urine are thawed at room temperature, then in 4 c with centrifugation 10 min of 10000 g to remove particulate matter；
B, pipette the urine specimen of 100 l with liquid-transfering gun, and add the pure water of 200 l, mix homogeneously, be then charged into chromatogram bottle
In to be measured；
C, reversed phase chromatography flight time mass spectrum are analyzed one by one to all urine specimens, obtain initial data；
D, convert raw data into the form of mzxml after, data is carried out with peak alignment, after correction, filter make an uproar, using simca-p
Carry out multivariate statistical analysis, the metabolite that screening vip is more than 1.
E, carry out t inspection with spss,p< 0.05 and vip be more than 1 for potential difference metabolite.
F, for potential difference metabolite, verified than counterpart method using library searching or standard substance, as poor
Different metabolite.
In step c, reverse phase liquid chromatography condition is: the acquity that all of sample is all produced using water company
(2.1 × 100 mm, 1.8 m) carry out separating uplc hss t3 post, and column temperature is 35 c, and mobile phase a is 0.1% formic acid
Aqueous solution, mobile phase b is 0.1% formic acid acetonitrile, and flow rate of mobile phase is 400 μ l/min, condition of gradient elution: 0-1 min, 2%
b-2%b；1-15 min, 2%b-45%b；15-16 min, 45%b-100%b；16-19 min, 100%b-100%b；19-20 min,
100%b-2%b, and maintain 2 min, the bulk analysis time is 22 min, and sample size is 5 μ l.
In step c, the condition of flight time mass spectrum detector: electric spray ion source, multiple-reaction monitoring cation scans
Mode；Spray voltage is 3500 v, and it is 4 l/min (180 DEG C) that gas is dried, and atomization gas are 0.04 mpa, and collision energy is 7
Ev, transmission time is 100 s.
The stability of the inventive method and repeatability:
Take the urine of equivalent, mix homogeneously, as matter from each laryngeal carcinoma volunteer's urine specimen and healthy volunteer's urine specimen
Control (qc) sample, adopt Quality Control (qc) sample come evaluation appts stability and repeatability to guarantee the reliability of experimental result,
Choose m/z respectively 180.0651,274.2731,320.1395,473.1515 and 551.3940 come evaluation appts stability and
Repeatability, the relative standard deviation (rsd) of this five endogenous Compound Retention time and peak area is shown in Table 1, retention time rsd
Less than 1%, peak area rsd is less than 20%, illustrates that the stability of system and repeatability all meet the requirements.
The stability of table 1 method and repeatability
The method of the present invention is a kind of discovery method of laryngeal carcinoma urine difference metabolite, optimizes pre-treatment side for urine specimen
Method, and the coherent detection condition of liquid chromatograph flight time mass spectrum is optimized, mainly optimize ion source condition, chromatograph
Post and flow visualizing.The inventive method has following excellent results:
1. liquid chromatograph flight time mass spectrum has the advantages that sensitivity height, high resolution.
2. this method pre-treatment is easy can improve analysis throughput it is not necessary to pass through complicated solid phase extraction procedure, tool
Have the advantages that quick and reproducible.
Brief description
Fig. 1. the chromatogram (b) of laryngeal carcinoma volunteer (a) and healthy volunteer's urine.
Fig. 2. the shot chart of laryngeal carcinoma volunteer's urine specimen and healthy volunteer's urine specimen, 1 is healthy person, and 2 is larynx
Cancer patient.
Specific embodiment
The present invention is described further below in conjunction with example, but is not to limit the present invention.
A kind of research method of the laryngeal carcinoma urine difference metabolite based on reversed phase chromatography flight time mass spectrum, its test process
To be urine be introduced directly into liquid chromatograph flight time mass spectrum after centrifugation, dilution is analyzed.
Example 1:
1. instrument and reagent: test the ultimate 3000 that used chromatograph of liquid is dionex, including liquid phase pump
(hpg-3400 sd), automatic sampler (wps-3000 sl) and column oven (tcc-3000 sd), all of sample all adopts
(2.1 × 100 mm, 1.8 m) carry out separating the acquity uplc hss t3 post that water company produces, and column temperature is 35
ºc.
Chromatographic grade formic acid, purity is 98%, buys from tedia company, acetonitrile, purity is 99.9%, buys from Korea S De Shan
Pharmaceutical industries strain (duksan pure chemivals), d- pantothenic acid, purity is more than 95%, buys from the easily safe scientific and technological limited public affairs in Wuhan
Shanghai branch company of department；Experimental water is deionized water.
2. sample treatment: urine is thawed at room temperature, urine in 4 c with centrifugation 10 min of 10000 g to remove
Particulate matter, then pipettes the urine specimen of 100 l with liquid-transfering gun, and adds the pure water of 200 l, mix homogeneously, is then charged into
To be measured in chromatogram bottle.
3. assay method: sample 5 l after absorption centrifugation, dilution, injection reversed phase chromatography flight time mass spectrum system is carried out
Separate and analyze.
4., after initial data is converted to the form of mzxml, peak identification, extraction and retention time correction are carried out to data,
Specific parameter is set to: the error that mass-to-charge ratio (m/z) allows is 10 ppm, and the width of chromatographic peak is 10-60 s, when reservation
Step-length be set to 1 m/z.
5. and then it is normalized using the data that simca-p 13.0 demo software extracts to xcms online, and
Carry out principal component analysiss, partial least squares discriminant analysis or orthogonal partial least squares discriminant analysis (opls-da), according to opls-
The result of da finds the change larger metabolite of ratio, then carries out t inspection using spss 20.0.
6. according to vip>1 and p<0.05 it is found that tens potential difference metabolite altogether, then utilize flight time matter
Spectrometer carries out secondary fragment scanning under mrm or automsn pattern, utilizes under conditions of allowable error scope is 10 ppm
Secondary fragment carries out library searching, so that it is determined that its why compound (metabolite).
Example 2: the method as described in embodiment 1, choose 37 laryngeal carcinoma volunteers and 29 healthy volunteer's urine specimens,
Identifying one of difference metabolite after data analysiss, library searching is d- pantothenic acid.
Claims (2)
1. a kind of mensure screening technique of the laryngeal carcinoma urine difference metabolite based on reversed phase chromatography flight time mass spectrum, its feature exists
In being to be measured for sample, compare, screen with laryngeal carcinoma volunteer and healthy volunteer's urine, specifically include following technique step
Rapid:
A, urine are thawed at room temperature, then in 4 c with centrifugation 10 min of 10000 g to remove particulate matter；
B, pipette the urine specimen of 100 l with liquid-transfering gun, and add the pure water of 200 l, mix homogeneously, be then charged into chromatogram bottle
In to be measured；
C, reversed phase chromatography flight time mass spectrum are analyzed one by one to all urine specimens, obtain initial data,
Reversed phase chromatography condition is: using acquity uplc hss t3 post specification 2.1 × 100 mm, 1.8 m are carried out point
From column temperature is 35 c, and mobile phase a is 0.1% aqueous formic acid, and mobile phase b is 0.1% formic acid acetonitrile, mobile phase stream
Speed is 400 μ l/min, gradient elution, and the bulk analysis time is 22 min, and sample size is 5 μ l；
The condition of flight time mass spectrum detector: electric spray ion source, multiple-reaction monitoring cation scan mode；Esi+ pattern:
Spray voltage is 3500 v, and it is 4 l/min (180 DEG C) that gas is dried, and atomization gas are 0.04 mpa, and collision energy is 7 ev, transmission
Time is 100 s；
D, convert raw data into the form of mzxml after, data is carried out with peak alignment, after correction, filter make an uproar, using simca-p
Multivariate data analysis software carries out multivariate statistical analysis, the metabolite that screening vip is more than 1；
E, carry out t inspection with spss, forp< 0.05 and vip be more than 1 for potential difference metabolite；
F, for potential difference metabolite, verified using library searching or standard substance comparison method, as difference metabolism
Thing.
2. according to claim 1 mensure screening technique it is characterised in that: the condition of gradient elution in step c is: 0-1
Min, 2%b-2%b；1-15 min, 2%b-45%b；15-16 min, 45%b-100%b；16-19 min, 100%b-100%b；19-
20 min, 100%b-2%b, and maintain 2 min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110687216A (en) * | 2018-07-06 | 2020-01-14 | 山西医科大学第一医院 | Application of endogenous serum micromolecules in evaluation of laryngeal cancer tumor cell differentiation degree |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102323351A (en) * | 2011-08-12 | 2012-01-18 | 深圳华大基因科技有限公司 | Bladder cancer patients urine specific metabolic thing spectrum, method for building up and purposes |
| US20120201747A1 (en) * | 2011-02-03 | 2012-08-09 | Pop Test Cortisol Llc | System and Method for Diagnosis and Treatment |
| WO2013162773A1 (en) * | 2012-04-27 | 2013-10-31 | Eastern Virginia Medical School | Quantitation of biomarkers for the detection of prostate cancer |
| CN104769112A (en) * | 2012-11-01 | 2015-07-08 | 菲克特生物科学股份有限公司 | Methods and products for expressing proteins in cells |
| CN105021804A (en) * | 2014-04-30 | 2015-11-04 | 湖州市中心医院 | Application of lung cancer metabolism markers to lung cancer diagnosis and treatment |
| CN105699514A (en) * | 2016-01-26 | 2016-06-22 | 上海交通大学 | Liquid chromatography-mass spectrometry model for detecting gastric cancer associated metabolism small molecule, and preparation method of liquid chromatography-mass spectrometry model |
| CN105738526A (en) * | 2016-03-11 | 2016-07-06 | 重庆市肿瘤研究所 | Method for screening specific serum metabolism markers for triple-negative breast cancer |
-
2016
- 2016-08-23 CN CN201610706956.2A patent/CN106370765A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120201747A1 (en) * | 2011-02-03 | 2012-08-09 | Pop Test Cortisol Llc | System and Method for Diagnosis and Treatment |
| CN102323351A (en) * | 2011-08-12 | 2012-01-18 | 深圳华大基因科技有限公司 | Bladder cancer patients urine specific metabolic thing spectrum, method for building up and purposes |
| WO2013162773A1 (en) * | 2012-04-27 | 2013-10-31 | Eastern Virginia Medical School | Quantitation of biomarkers for the detection of prostate cancer |
| CN104769112A (en) * | 2012-11-01 | 2015-07-08 | 菲克特生物科学股份有限公司 | Methods and products for expressing proteins in cells |
| CN105021804A (en) * | 2014-04-30 | 2015-11-04 | 湖州市中心医院 | Application of lung cancer metabolism markers to lung cancer diagnosis and treatment |
| CN105699514A (en) * | 2016-01-26 | 2016-06-22 | 上海交通大学 | Liquid chromatography-mass spectrometry model for detecting gastric cancer associated metabolism small molecule, and preparation method of liquid chromatography-mass spectrometry model |
| CN105738526A (en) * | 2016-03-11 | 2016-07-06 | 重庆市肿瘤研究所 | Method for screening specific serum metabolism markers for triple-negative breast cancer |
Non-Patent Citations (3)
| Title |
|---|
| 和红兵 等: "气相色谱-质谱和液相色谱-质谱联用方法用于口腔癌代谢组学分析", 《色谱》 * |
| 张小涛 等: "《中国化学会第二届全国质谱分析学术报告会会议论文集》", 31 October 2015 * |
| 汪江山 等: "超高效液相色谱/飞行时间质谱用于人参皂甙Rg3作用后大鼠尿液代谢物指纹图谱分析及标记物的鉴定", 《色谱》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110687216A (en) * | 2018-07-06 | 2020-01-14 | 山西医科大学第一医院 | Application of endogenous serum micromolecules in evaluation of laryngeal cancer tumor cell differentiation degree |
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Application publication date: 20170201 |