CN110470764A - A kind of semi-quantitative analysis method of lipid - Google Patents
A kind of semi-quantitative analysis method of lipid Download PDFInfo
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Abstract
Technical solution of the present invention discloses a kind of semi-quantitative analysis method of lipid, by sample to be tested, internal standard reagent, extract reagent and separation agent after mixing, its layering is set to form upper layer sample to be tested and lower layer's sample to be tested, pass through high performance liquid chromatography-tandem mass system detection lower layer's sample to be tested and calibration reagent, to obtain the Mass Spectrometer Method of calibration reagent and sample to be tested as a result, obtaining the semi-quantitative results of lipid in sample to be tested referring to internal standard.The analysis method of technical solution of the present invention effectively can carry out semi-quantitative analysis to lipid using a small amount of serum.
Description
Technical field
The invention belongs to the analysis technical fields of compound, and in particular to a kind of semi-quantitative analysis method of lipid.
Background technique
Blood is made of cell and body fluid components, includes red blood cell, leucocyte and blood platelet in cell component, body fluid is blood
Starching (serum) includes the various protein with specific function and numerous small molecule metabolite.Blood follows in vivo
Ring is reciprocal, and interdependence is respectively organized with human body, is influenced each other, the metabolic alterations of body physiological pathology are able to reflect, and leads to these
The detection and analysis of metabolite, nuance, feature and satellite phenomenon between understanding, it will be able to be provided for us important
Clue or experimental basis.Amino acid, fatty acyl carnitine and fatty acid are metabolites important in blood.
Lipid metabolism product in serum is the important indicator in plasma metabolism.Due to the water solubility of this kind of metabolite
It is not strong.Therefore, using conventional chromatograph-mass spectrometer coupling system, it is difficult to accomplish effective, reliable, high specific quantitative analysis.
Therefore, under the premise of guaranteeing specificity and quantitative detection, the content for the lipoid substance how to be effectively detected,
It is difficulties urgently to be resolved.
Summary of the invention
Technical solution of the present invention technical problems to be solved are that existing analysis method is difficult to accomplish effective, reliable, Gao Te
Anisotropic analysis lipid material.
In order to solve the above technical problems, technical solution of the present invention provides a kind of semi-quantitative analysis method of lipid, Ke Yiyou
The content of the lipoid substance of the detection of effect.It even can be under the premise of guaranteeing specificity and quantitative performance, using minute quantity
Serum (such as 10 μ L serum) can effectively analyze the content of lipoid substance.It includes the following steps:
(1) sample to be tested is collected；
(2) internal standard reagent is prepared respectively, extracts reagent and separation agent；
(3) sample to be tested, the internal standard reagent, the extraction reagent and the separation agent are mixed by a certain percentage
After closing uniformly, its layering is made to form upper layer sample to be tested and lower layer's sample to be tested；
(4) lower layer's sample to be tested described in extraction section, and lower layer's sample to be tested of extraction is subjected to high performance liquid chromatography-string
Join mass spectrometer system detection；
(5) mass spectrogram for obtaining internal standard reagent and sample to be tested, obtains the sxemiquantitative of lipid in sample to be tested referring to internal standard
As a result.
Preferably, the sample to be tested is test serum；The serum sample can be obtained from blood sample.
Preferably, the internal standard reagent includes d14:1 phosphatidyl choline, d18:1-12:0 sphingomyelins, D7-d15:0 cholesterol
Ester, d15:0 phosphatidyl ethanolamine, d15:0 phosphatide glycerol, d18:1-17:0 ceramide, d18:1-12:0 galactoside nerve acyl
Amine, d17:0 phosphatidic acid, D7- cholesterol, d18:1-24:0 ceramide phosphate and D5-17:0-17:1-17:0 triglycerides
At least one of.
Preferably, the extraction reagent includes chloroform and/or methanol.
Preferably, the separation agent includes lithium chloride and/or water.
Preferably, lower layer's sample to be tested by extraction carries out high performance liquid chromatography-tandem mass system detection, including
Following steps:
Highly effective liquid phase chromatographic system is balanced using Mobile-phase reagent；
Remove all chromatographic columns in highly effective liquid phase chromatographic system；
Lower layer's sample to be tested of extraction is injected into high performance liquid chromatography-tandem mass system in a manner of flow injection sample introduction
In tested and analyzed.
Preferably, the Mobile-phase reagent includes the first Mobile-phase reagent, the second Mobile-phase reagent and the examination of third mobile phase
Agent；
First Mobile-phase reagent is the methanol solution of lithium hydroxide, is used for phosphatidyl choline, sphingomyelins and galactoside
The quantitative analysis of ceramide；
Second Mobile-phase reagent is the methanol solution of ammonium hydrogen carbonate, is used for ceramide, ceramide phosphate, phosphorus
The quantitative analysis of lipsitol, phosphatidic acid, phosphatide glycerol and phosphatidyl ethanolamine；
The third Mobile-phase reagent is the methanol solution of ammonium formate and formic acid, for cholesteryl ester, diglyceride and sweet
The quantitative analysis of oily three esters.
Preferably, in highly effective liquid phase chromatographic system, respectively using the first Mobile-phase reagent, the second Mobile-phase reagent and the
Three Mobile-phase reagents separate lipid with arbitrary serial order, and separating obtained sample to be tested is as a batch every time
Tandem mass spectrum system is added.
It is preferably, described to obtain the semi-quantitative results of lipid in sample to be tested referring to internal standard, comprising:
According to the mass spectrogram of internal standard reagent and sample to be tested, the opposite peak mass spectrogram of internal standard reagent and sample to be tested is obtained；
In the opposite peak mass spectrogram of internal standard reagent and sample to be tested, corresponding lipid in internal standard reagent and sample to be tested is obtained
The ratio between peak value, the ratio of number of corresponding lipid is equal to according to the ratio between peak value of corresponding lipid, calculates the quantity of corresponding lipid.
Preferably, the tandem mass spectrum system uses electric spray ion source and/or atmospheric chemical ionization ion source.
Preferably, for phosphatidyl choline, sphingomyelins, galactosyl ceramide, ceramide, ceramide phosphate,
Phospholipid inositol, phosphatidic acid, phosphatide glycerol and phosphatidyl ethanolamine quantitative analysis when, use electric spray ion source；For cholesterol
When the quantitative analysis of ester, diglyceride and triglycerides, atmospheric chemical ionization ion source is used.
Preferably, the tandem mass spectrum system is quadrupole rod tandem mass spectrum system.It is further preferred that being three level four bars
The tandem mass spectrum system that mass spectrograph is connected in series.
Preferably, the detection method of the tandem mass spectrum system be more reaction channels detection, including precursor scans, son from
At least two in son scanning and neutral loss scan.
Preferably, it in step (3), is layered using centrifugal separation, centrifugal force is 10000g~12500g, time
For 5~10min.
Preferably, the mixing method be liquid-transfering device blow and beat, be mixed by inversion, rotate mix and/or vortex oscillation mix
It is even, it is therefore preferable to which that vortex oscillation mixes 1~5min.
Compared with prior art, technical solution of the present invention has the advantages that the flowing by high performance liquid chromatography
Chromatographic column separating step is omitted in injection, and the consumption of sample can be effectively reduced, reduce the time of analysis；By successively using not homopolarity
Property mobile phase solve lipoid substance in serum so that the unsuitable substance of dissolubility is separated in chromatographic system in batches
Dissolubility and the unmatched problem of conventional chromatogram system；Guarantee the stability and reproducibility of analysis by the way that internal standard is added；Through liquid phase
Analyte importing tandem mass spectrum is detected, can effectively ensure that detection sensitivity and specificity；Joined by liquid chromatography mass spectrometric
With analysis；The content of a variety of lipoid substances can be detected simultaneously under the premise of guaranteeing detection accuracy and stability, improve and divide
The flux of analysis.
Detailed description of the invention
Fig. 1 is the mass spectrogram of ceramide phosphate (d18:1-16:0) in the embodiment of the present invention；
Fig. 2 is the mass spectrogram of phosphatidylinositols (d18:0-22:6) in the embodiment of the present invention；
Fig. 3 is the mass spectrogram of cholesterol (d14:0CE) in the embodiment of the present invention.
Specific embodiment
In order to make art technology field personnel more fully understand the technical solution in the application, below in conjunction with following knot
Closing embodiment, the invention will be further described, it is clear that and described embodiments are only a part of embodiments of the present application, without
It is whole embodiments.Based on the embodiment in the application, those of ordinary skill in the art are not before making creative work
All other embodiment obtained is put, shall fall within the protection scope of the present application.It is right with reference to the accompanying drawings and embodiments
The present invention is further described.
The embodiment of the present invention makees the semi-quantitative analysis method of technical solution of the present invention for analyzing the lipid in serum
To be described in detail.Certainly, in addition to detecting serum, the detection method of technical solution of the present invention can also be applied to other and need to detect
In the fluid environment of lipid.
The semi-quantitative analysis method of the lipid of the embodiment of the present invention, comprising the following steps:
(1) sample to be tested is collected: acquisition 10 μ L of serum.
(2) internal standard reagent is prepared respectively, extracts reagent and separation agent.Ingredient and concentration (or the body of above-mentioned three kinds of reagents
Product) as shown in table 1-3.
The ingredient and concentration of 1 internal standard reagent of table
Note: solvent is methanol.
The ingredient and volume of the extraction reagent of table 2
The ingredient and concentration of 3 separation agent of table
(3) sample to be tested, the internal standard reagent, the extraction reagent and the separation agent are mixed by a certain percentage
After closing uniformly, its layering is made to form upper layer sample to be tested and lower layer's sample to be tested.Specifically, by sample to be tested, internal standard reagent, mention
Take reagent and separation agent by the volume mixture of table 4, and vortex oscillation 1min is to mix well.
The mixed volume of 4 serum of table and classes of agents
By the sample after being mixed evenly under the centrifugal force of 12000g, it is centrifuged 5min, sample is divided into upper and lower two after centrifugation
Layer, there is apparent interface therebetween.
(4) lower layer's sample to be tested described in extraction section, and lower layer's sample to be tested of extraction is subjected to high performance liquid chromatography-string
Join mass spectrometer system detection.
Firstly, three kinds of Mobile-phase reagents (A, B, C) are successively used, it is efficient with the flow velocity balance Vanquish of 3.00mL/min
Liquid chromatogram 5min, the specific ingredient and concentration of Mobile-phase reagent (A, B, C) are as shown in table 5~7.
The ingredient and concentration of 5 Mobile-phase reagent A of table
The ingredient and concentration of 6 Mobile-phase reagent B of table
The ingredient and concentration of 7 Mobile-phase reagent C of table
Then all chromatographic columns in highly effective liquid phase chromatographic system are removed.
Sample introduction successively is implemented to six kinds of calibration reagents from low to high by concentration；
Lower layer's sample to be tested of extraction is injected into high performance liquid chromatography-tandem mass system in a manner of flow injection sample introduction
In tested and analyzed.
Above-mentioned mobile phase of high performance liquid chromatography and separation condition are as shown in table 8.
8 mobile phase of high performance liquid chromatography of table and separation condition
Reaction detection scan pattern is carried out through a quadrupole rods tandem mass spectrometry combined system after.Specifically, for preceding two
The outlet of high performance liquid chromatography is connected in the electric spray ion source of mass spectrometer system by sample after the separation that a batch obtains, and is used
In phosphatidyl choline, sphingomyelins, galactosyl ceramide, ceramide, ceramide phosphate, phospholipid inositol, phosphatidic acid, phosphorus
The semi-quantitative analysis of the lipid materials such as rouge glycerol and phosphatidyl ethanolamine；Sample after the separation obtained for the last one batch, will
The outlet of high performance liquid chromatography is connected in mass spectrographic normal pressure chemical ion source, is used for cholesteryl ester, diglyceride and glycerol three
The semi-quantitative analysis of the substances such as ester.
Wherein mass spectrographic testing conditions are as follows:
Phosphatidyl choline, sphingomyelins and galactosyl ceramide:
Ionization mode: electrospray ionisation, ESI (+)；
Ion spray voltage: 3.5kV；
Sheath gas (Arb): 20；
Assistor (Arb): 5；
Purge gass (Arb): 0；
Ion transfer capillary temperature (DEG C): 300；
Sprayer temperature (DEG C): 150；
Detection mode: Selective reaction monitoring (SRM)；
Circulation time (sec): 1.2；
Q1 resolution ratio (FWHM): 0.7；
Q3 resolution ratio (FWHM): 0.7；
Collision gas (mTorr): 1.5；
Voltage (v) is cracked in source: 0；
Ceramide, ceramide phosphate, phospholipid inositol, phosphatidic acid, phosphatide glycerol and phosphatidyl ethanolamine:
Ionization mode: electrospray ionisation, ESI (-)；
Ion spray voltage: 3.5kV；
Sheath gas (Arb): 20；
Assistor (Arb): 5；
Purge gass (Arb): 0；
Ion transfer capillary temperature (DEG C): 300；
Sprayer temperature (DEG C): 150；
Detection mode: Selective reaction monitoring (SRM)；
Circulation time (sec): 1；
Q1 resolution ratio (FWHM): 0.7；
Q3 resolution ratio (FWHM): 0.7；
Collision gas (mTorr): 1.5；
Voltage (v) is cracked in source: 0；
Cholesteryl ester, diglyceride and triglycerides:
Ionization mode: atmosphere pressure chemical ion source, APCI (+)；
Cation exchanging electric current (uA): 8；
Cation exchanging electric current (uA): 10；
Sheath gas (Arb): 20；
Assistor (Arb): 5；
Purge gass (Arb): 0；
Ion transfer capillary temperature (DEG C): 300；
Sprayer temperature (DEG C): 300；
Detection mode: Selective reaction monitoring (SRM)
Circulation time (sec): 1
Q1 resolution ratio (FWHM): 0.7
Q3 resolution ratio (FWHM): 0.7
Collision gas (mTorr): 1.5
Voltage (v) is cracked in source: 0
(5) mass spectrogram for obtaining internal standard reagent and sample to be tested, obtains the sxemiquantitative of lipid in sample to be tested referring to internal standard
As a result.
Since the embodiment of the present invention can detecte out the lipid material compared with multiple types, therefore Fig. 1~3 illustrate only wherein three kinds
The mass spectrogram of lipid material, and signal strength and channel information are shown in figure.
Relative quantification calculation is finally used, according to the ratio of the peak area of the peak area of each test substance and internal standard reagent
Value (Area Ratio) and the product of the internal standard reagent concentration obtain the concentration of test substance, the substance as in test serum
Content.
Although the present invention discloses as above in a preferred embodiment thereof, it is not for limiting the present invention, any ability
Field technique personnel without departing from the spirit and scope of the present invention, may be by the methods and technical content of the disclosure above to this
Inventive technique scheme makes possible variation and modification, therefore, anything that does not depart from the technical scheme of the invention, according to this hair
Bright technical spirit belongs to the technology of the present invention to any simple modifications, equivalents, and modifications made by embodiment of above
The protection scope of scheme.
Claims (10)
1. a kind of semi-quantitative analysis method of lipid, which comprises the steps of:
(1) sample to be tested is collected；
(2) internal standard reagent is prepared respectively, extracts reagent and separation agent；
(3) sample to be tested, the internal standard reagent, the extraction reagent and the separation agent are mixed by a certain percentage equal
After even, its layering is made to form upper layer sample to be tested and lower layer's sample to be tested；
(4) lower layer's sample to be tested described in extraction section, and lower layer's sample to be tested of extraction is subjected to high performance liquid chromatography-series connection matter
Spectra system detection；
(5) mass spectrogram for obtaining internal standard reagent and sample to be tested, obtains the semi-quantitative results of lipid in sample to be tested referring to internal standard.
2. the semi-quantitative analysis method of lipid as described in claim 1, which is characterized in that the sample to be tested is blood to be measured
Clearly.
3. the semi-quantitative analysis method of lipid as described in claim 1, which is characterized in that the internal standard reagent includes d14:1
Phosphatidyl choline, d18:1-12:0 sphingomyelins, D7-d15:0 cholesteryl ester, d15:0 phosphatidyl ethanolamine, d15:0 phosphatide glycerol, d18:
1-17:0 ceramide, d18:1-12:0 galactosyl ceramide, d17:0 phosphatidic acid, D7- cholesterol, d18:1-24:0 mind
Through at least one of amidophosphoric acid ester and D5-17:0-17:1-17:0 triglycerides.
4. the semi-quantitative analysis method of lipid as described in claim 1, which is characterized in that the extraction reagent includes chloroform
And/or methanol；Preferably, the separation agent includes lithium chloride and/or water.
5. the semi-quantitative analysis method of lipid as described in claim 1, which is characterized in that described that the lower layer of extraction is waited for test sample
This progress high performance liquid chromatography-tandem mass system detection, includes the following steps:
Highly effective liquid phase chromatographic system is balanced using Mobile-phase reagent；
Remove all chromatographic columns in highly effective liquid phase chromatographic system；
By lower layer's sample to be tested of extraction injected in a manner of flow injection sample introduction in high performance liquid chromatography-tandem mass system into
Row tests and analyzes.
6. the semi-quantitative analysis method of lipid as claimed in claim 5, which is characterized in that the Mobile-phase reagent includes first
Mobile-phase reagent, the second Mobile-phase reagent and third Mobile-phase reagent；
First Mobile-phase reagent is the methanol solution of lithium hydroxide, for phosphatidyl choline, sphingomyelins and galactoside nerve
The quantitative analysis of amide；
Second Mobile-phase reagent is the methanol solution of ammonium hydrogen carbonate, is used for ceramide, ceramide phosphate, phosphatide flesh
The quantitative analysis of alcohol, phosphatidic acid, phosphatide glycerol and phosphatidyl ethanolamine；
The third Mobile-phase reagent is the methanol solution of ammonium formate and formic acid, is used for cholesteryl ester, diglyceride and glycerol three
The quantitative analysis of ester.
7. the semi-quantitative analysis method of lipid as claimed in claim 6, which is characterized in that in highly effective liquid phase chromatographic system,
Respectively using the first Mobile-phase reagent, the second Mobile-phase reagent and third Mobile-phase reagent with arbitrary serial order to lipid into
Row separation, tandem mass spectrum system is added as a batch in separating obtained sample to be tested every time.
8. the semi-quantitative analysis method of lipid as described in claim 1, which is characterized in that described to obtain referring to internal standard to test sample
The semi-quantitative results of lipid in this, comprising:
According to the mass spectrogram of internal standard reagent and sample to be tested, the opposite peak mass spectrogram of internal standard reagent and sample to be tested is obtained；
In the opposite peak mass spectrogram of internal standard reagent and sample to be tested, the peak of corresponding lipid in internal standard reagent and sample to be tested is obtained
The ratio between value is equal to the ratio of number of corresponding lipid according to the ratio between peak value of corresponding lipid, calculates the quantity of corresponding lipid.
9. the semi-quantitative analysis method of lipid as described in claim 1, which is characterized in that the tandem mass spectrum system uses electricity
Esi ion source and/or atmospheric chemical ionization ion source.
10. the semi-quantitative analysis method of lipid as claimed in claim 9, which is characterized in that for phosphatidyl choline, sphingomyelins,
Galactosyl ceramide, ceramide, ceramide phosphate, phospholipid inositol, phosphatidic acid, phosphatide glycerol and phosphatidyl ethanolamine
Quantitative analysis when, use electric spray ion source；When quantitative analysis for cholesteryl ester, diglyceride and triglycerides, make
With atmospheric chemical ionization ion source.
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