CN106153763B - The hydrophilic chromatographic of phosphatide-tandem mass spectrum detection method in the new prawn of knife volume - Google Patents
The hydrophilic chromatographic of phosphatide-tandem mass spectrum detection method in the new prawn of knife volume Download PDFInfo
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- 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
- G01N30/02—Column chromatography
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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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
, sample pre-treatments the invention discloses a kind of hydrophilic chromatographic-tandem mass spectrum detection methods of phosphatide in new prawn of knife volume, comprising the following steps: 1): by knife volume newly to shrimp be ground into shrimp it is rotten after carry out corresponding pre-treatment, obtain lipid crude extract；2) lipid crude extract, liquid phase separation: is subjected to liquid phase separation；Chromatographic column is YMC Triart glycol-based HILIC column；Chromatographic condition uses linear gradient elution method, and gradient system is made of mobile phase A and Mobile phase B；Wherein mobile phase A is the acetonitrile solution containing 53mmol/L formic acid, and Mobile phase B is the aqueous solution containing 60mM ammonium formate and 53mM formic acid；3) the resulting eluent of step 2), is subjected to Mass Spectrometer Method analysis.The total content of PE, PS and PC in sample can be accurately detected using method of the invention, and realize and identification is analyzed by mass spectrometry to each molecular species in PC, PE and PS three classes phosphatide.
Description
Technical field
The invention belongs to field of food detection, it is related to a kind of hydrophilic chromatographic-tandem mass spectrum inspection of phosphatide in the new prawn of knife volume
Survey method refers specifically to phosphatidyl choline in a kind of new prawn of knife volume, phosphatidyl-ethanolamine, phosphatidylserine three categories phosphatide
Structural Identification and quantitative analysis method.
Background technique
The new prawn of knife volume (Metapenaeus ensis), i.e. shrimp are that China cultivates one of small-sized marine products shrimps.Cause
It has many advantages, such as that feeding habits are miscellaneous, growth is fast, high temperature resistant, proper salinity level is wide, anti-poor environment ability is strong, in recent years Central-South coastal
Artificial breeding has been carried out extensively in area, shows good economic prospect.Shrimps aquatic resources lipid content is high, is rich in Ω -3 race
With Ω -6 race polyunsaturated fatty acid and phosphatidyl choline (Phosphatidylcholine, PC), phosphatidyl-ethanolamine
The phospholipid substances such as (Phosphatidylethanolamine, PE), phosphatidylserine (Phosphatidlserine, PS).
Phosphatide is a kind of lipid containing phosphoric acid.Structure of phospholipid mainly by glycerol backbone, polar group and different length and
The fatty acid chain of saturation degree forms.It is different according to phosphatide polar group, phosphatide can be divided into phosphatidyl choline, phosphatidyl ethanol
The classifications such as amine, phosphatidylserine.Phosphatide kind present in organism is thousands of, and various structures, type are complicated, has uniqueness
Chemical structure.Phosphatide is the cyto-architectural important component of maintenance, and the nutrient that metabolism is indispensable.Phosphorus
Rouge is also equipped with extensive biological function, such as activating cell, and maintenance metabolism is balanced with hormone secretion, enhances human immunity
Deng.It is demonstrated experimentally that phosphatide can reduce blood lipid, cholesterol, prevents superabundant fats from depositing in vascular wall, keeps vascular circulation smooth,
It is known as " blood vessel scavenger ".The detection method of phospholipid molecule is beneficial to the in-depth new prawn of knife volume in the new prawn of efficient knife volume
The Nutritional studies of middle phosphatide, promoting the new prawn of knife volume is the product development of raw material.
Traditional phosphatide detection method includes high performance liquid chromatography (HPLC), NMR spectrum (NMR), fluorescence spectrum
(FLR) etc., time-consuming, sensitivity is low for the analysis of these methods.Hydrophilic chromatographic (HILIC) is specially to separate highly polar and hydrophily
Close a kind of method of object.Because making hydrophilic chromatographic that there is preferable mass spectrum using conventional reverse-phase chromatography mobile phase.
The conventional method of existing lipid separation at present is normal-phase chromatography, need special chromatographic equipment and mobile phase (just oneself
Alkane, isopropanol, chloroform), and the resulting eluent of the mobile phase can not be directly combined with mass spectroscopy device.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of easy to operate, stronger new prawns of knife volume of qualitative, quantitative ability
The hydrophilic chromatographic of middle phosphatidyl choline, phosphatidyl-ethanolamine and phosphatidylserine-tandem mass spectrum detection method.
In order to solve the above technical problem, the present invention provides a kind of hydrophilic chromatographic-series connection matter of phosphatide in new prawn of knife volume
Spectrum detection method, comprising the following steps:
1), sample pre-treatments:
Knife volume is newly ground into shrimp gruel to shrimp, according to the solid-liquid ratio of 1g/15~20ml, organic solvent is added in shrimp gruel
It shakes and mixes after I；Then it carries out ultrasonic (probe type ultrasonic) ice bath and extracts (extraction time is 10~15 minutes), after extraction
Pure water is added and shakes mixing, obtains mixture, the ultrapure water of 10~15mL of every 1g shrimp gruel adapted；Organic solvent I is methanol and chlorine
The volume ratio of imitative mixed liquor, methanol and chloroform is 0.5~2:1；
Mixture is centrifuged with refrigerated centrifuge, pipettes lower phase solution from centrifugation gains；The lower phase solution low temperature (≤
10 DEG C) after dry (carrying out low temperature drying with nitrogen), with the redissolution of organic solvent II, obtain lipid crude extract；The organic solvent II
The methanol aqueous solution or methanol for being volumetric concentration >=30% are (that is, 30~100% methanol aqueous solution, preferably 90% methanol
Aqueous solution),
2), liquid phase separation:
Lipid crude extract is subjected to liquid phase separation；Chromatographic column be YMC Triart glycol-based HILIC column (4.6 × 250mm, 3
μm).Chromatographic condition uses linear gradient elution method, and gradient system is made of mobile phase A and Mobile phase B；Wherein mobile phase A be containing
The acetonitrile solution (pH is about 4.0~4.5) of 53mmol/L formic acid, Mobile phase B are the water containing 60mM ammonium formate and 53mM formic acid
Solution (pH is about 3.6)；
Gradient system are as follows: 0~15min maintains 2% mobile phase A；Mobile phase A is increased to by 15~60min from 2%
40%；60~70min maintains 40% mobile phase A；Mobile phase A is dropped back to the 2% of original state from 40% by 70~80min；On
Stating % is volume %；
Remarks explanation: the eluent of liquid phase is directly entered mass spectral analysis；
Liquid chromatogram uses 1100 system of Agilent, is equipped with the members such as quaternary pump, autosampler, chromatographic column insulating box
Part；
3) the resulting eluent of step 2), is subjected to Mass Spectrometer Method analysis.
Hydrophilic chromatographic-tandem mass spectrum detection method improvement as phosphatide in the new prawn of knife volume of the invention:
The step 3):
Using QSTAR level four bars/time of-flight mass spectrometer, it is equipped with TurboIonSpray ion source；Negative ion mode inspection
It surveys；Scanning range is 500~1000Da；Removing cluster voltage (DP) is 50V；Focusing potential (FP) is 200V；Ion source voltage is
4500V；Removing cluster potential 2 (DP2) is 10V；Gas source 1 (GS1) is 50psi；Gas source 2 (GS2) is 45psi；Gas curtain gas (CUR) is
40psi；Temperature is 400 DEG C；The impact energy that daughter ion scanning (PIS) uses is 20V；Data collection and analysis uses Analyst
QS v2.0。
Remarks explanation: liquid chromatogram can be separated phosphatide by class, and such as Fig. 1, each peak represents a kind of phosphatide, and (some peaks are miscellaneous
Matter), it is different from double bond quantity according to carbon chain lengths difference in every class phosphatide, it include many phospholipid molecules, mass spectrum is to every class phosphatide
After detection, such as Fig. 2, the mass-to-charge ratioes of the different phospholipid molecules of available every class phosphatide (i.e. the ratio between quality and charge, in this experiment
Charge is 1, so the value of mass-to-charge ratio is exactly phospholipid molecule quality), each peak represents a phospholipid molecule kind, according to Phospholipids
Amount, searching library using lipidview just can be obtained structural information.
Hydrophilic chromatographic-tandem mass spectrum detection method further improvement as phosphatide in the new prawn of knife volume of the invention:
In the step 1), to pipette the centrifugation gains after lower phase solution (that is, including upper phase solution, positioned above mixing
Solids between liquid and lower phase solution) substitution shrimp gruel, organic solvent I is substituted with the chloroform in organic solvent I, repeats progress
It extracts 1~3 time；
Merge after the resulting lower phase solution of all extraction steps merges and carries out subsequent drying and redissolution.
Remarks explanation: lower leaf on solution in centrifuge tube after centrifugation, knife volume are newly then in that pie occupy in two-phase to shrimp meat
Between.
Methanol, chloroform, water three together when will not be miscible, two-phase up and down can be divided into, methanol polarity more by force can be with
Water is miscible, therefore in centrifuge tube, and the miscible a small amount of methanol of chloroform is in lower phase, and the miscible volume methanol of water is in upper phase；Due to pipetting away
After lower phase, practical major part methanol is not pipetted in upper phase still, therefore repeats when extraction only to need that chloroform is added
?.That is, the volumetric usage of chloroform when repeating to extract in volumetric usage=solvent I of chloroform.
Hydrophilic chromatographic-tandem mass spectrum detection method further improvement as phosphatide in the new prawn of knife volume of the invention:
Ultrasound condition is 25kHz in the step 1).
Hydrophilic chromatographic-tandem mass spectrum detection method further improvement as phosphatide in the new prawn of knife volume of the invention:
Refrigerated centrifuge is in 4 DEG C with 8000rpm centrifugation 15 minutes in the step 1).
Hydrophilic chromatographic-tandem mass spectrum detection method further improvement as phosphatide in the new prawn of knife volume of the invention:
In the step 2), flow velocity when gradient elution is 180~220 μ L/min (preferably 200 μ L/min).
Hydrophilic chromatographic-tandem mass spectrum detection method further improvement as phosphatide in the new prawn of knife volume of the invention:
Sample volume (that is, amount of lipid crude extract) is 5~10 μ L in the step 2).
Hydrophilic chromatographic-tandem mass spectrum detection method further improvement as phosphatide in the new prawn of knife volume of the invention:
In the step 1), the solid-liquid ratio of the organic solvent II of shrimp gruel and redissolution is 1g/1~2ml.
In the sample pre-treatments of step 1) of the invention:
The new prawn muscle tissue sample of knife volume (shrimp is rotten) after taking homogeneous is in centrifuge tube, and concussion is mixed after organic solvent I is added
It is even.Probe type ultrasonic ice bath extracts, and pure water is added to centrifuge tube after finishing and shakes mixing.Mixture refrigerated centrifuge from
The heart, lower leaf on solution in centrifuge tube after centrifugation, the new prawn muscle tissue sample of knife volume are then in that pie occupy among two-phase.With shifting
Lower phase solution is transferred to another new pipette by liquid rifle, is continued up mutually and is added in sample chloroform and repeat aforesaid operations
Extract 1~3 time (for example, twice).The organic phase of extraction is merged, is dried up with nitrogen low temperature, and redissolved with methanol aqueous solution.
In the present invention, the PTFE filter membrane that 0.22 μm is crossed after redissolution, obtains lipid crude extract.
The present invention uses glycol-based HILIC column, using the acetonitrile solution of 53mmol/L formic acid as mobile phase A, 60mM ammonium formate
Aqueous solution with 53mM formic acid is Mobile phase B；(0~15min maintains 2% mobile phase A after gradient elution；15~60min, will
Mobile phase A is increased to 40% from 2%；60~70min maintains 40% mobile phase A；70~80min drops mobile phase A from 40%
Return the 2% of original state) phosphatide is separated.Compared to conventional chromatogram technology, this method can make lipid separation in laboratory routine instrument
Accomplished in device reverse-phase chromatography, equipment needed for effectively preventing traditional normal-phase chromatography and mobile phase use methanol or acetonitrile
, and can be directly combined with mass spectrum.
Detection method sample preparation of the invention, liquid phase separation, Mass Spectrometer Method, it is easy to operate, simple easily training, qualitative fixed
Amount ability is stronger, practical, and suitable laboratory is widely used.
In conclusion in a kind of new prawn of knife volume that the present invention establishes phosphatide hydrophilic chromatographic-tandem mass spectrum detection method
Simple and quick, high sensitivity is as a result reliable and stable, is of great significance for the development of aquatic products iipidomic.Using this
The method of invention can accurately detect the total content of PE, PS and PC in sample, and realize to each in PC, PE and PS three classes phosphatide
A molecular species are analyzed by mass spectrometry identification.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is that the HILIC-TOF/MS of the new prawn musculature lipid-soluble extract of knife volume detects figure；
TIC of+TOF MS:from Sample of 20150610.wiff (Turbo Spray).
The mass spectrogram of PC, PE and PS in the new prawn lipid-soluble extract of the position Fig. 2 knife volume；
Upper figure (PC):
+TOF MS:20.176 to21.528min from Sample7of 20150610.wiff
A=3.59917355969297020e-004, t0=-5.28098098505142840e+000 (Turbo
Spray)
Middle figure (PE):
+TOFMS:49.449to50.000min from Sample7of 20150610.wiff
A=3.59917355969297020e-004, t0=-5.28098098505142840e+000 (Turbo
Spray)
The following figure (PS):
+ TOFMS:48.899to49.449min from Sample7of 20150610.wiff
A=3.59917355969297020e-004, t0=-5.28098098505142840e+000 (Turbo
Spray)。
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This.
In the present invention:
Material and reagent
Three kinds of phosphatide standard items: PC-14:0/14:0, PE-15:0/15:0 and PS-14:0/14:0 purity are all larger than
99.9% (Avanti Polar Lipids company of the U.S.)；The standard items mother liquor of 1mg/mL is configured to using methanol as solvent.Chlorine
The reagent purchases such as imitative, methanol, acetonitrile are from Merck company of Germany.The ultrapure water that resistivity is 18.2M Ω cm is filtered from Milli-
Q system (Millipore company of the U.S.).
Hydrophilic chromatographic-tandem mass spectrum detection method of phosphatide in embodiment 1, a kind of new prawn of knife volume
Specially successively follow the steps below:
1), sample pre-treatments:
After the new prawn of knife volume removes excretory gland, abdomen band musculature is taken as shrimp and is ground into the rotten (slurry of shrimp
Shape).The gruel of 1.0g shrimp is weighed in 5mL centrifuge tube, 18mL chloroform/methanol mixed liquor (1:2, v/v) is added, and concussion mixes afterwards.In 250W
Lower ultrasonic wave (25kHz) ice bath, which extracts, to be added 12.5mL ultrapure water to centrifuge tube after ten minutes and shakes mixing.Mixture is with cold
Freeze centrifuge (Thermo Scientific company of the U.S.) in 4 DEG C with 8000g centrifugation 5 minutes, after centrifugation in centrifuge tube on solution
Lower leaf, tissue sample are then in that pie occupy among two-phase.Lower phase solution is transferred to another new pipette with liquid-transfering gun,
It continues up mutually and 6mL chloroform is added in sample and repeats aforesaid operations and extract twice.By the organic phase extracted three times (under mix
Liquid) merge, it is dried up with nitrogen low temperature, and cross 0.22 μm after being redissolved with methanol aqueous solution 2ml (methanol: water=9:1, v/v)
PTFE filter membrane.
2), liquid phase separation
Liquid chromatogram experiment uses 1100 system of Agilent, is equipped with quaternary pump, autosampler, chromatographic column insulating box etc.
Element.Chromatographic column is YMC Triart glycol-based HILIC column (4.6 × 250mm, 3 μm).Chromatographic condition uses linear gradient elution method,
Wherein mobile phase A is the acetonitrile solution (pH is about 4.0~4.5) containing 53mM formic acid, and Mobile phase B is the ammonium formate containing 60mM
With the aqueous solution of 53mM formic acid (pH is about 3.6).
Gradient system is made of mobile phase A and Mobile phase B；Gradient system is as follows: 0~15min, maintains 2% mobile phase A；
Mobile phase A is increased to 40% from 2% by 15~60min；60~70min maintains 40% mobile phase A；70~80min will flow
Phase A drops back to original state, i.e., 2% from 40%.Above-mentioned % is volume %.The flow control of gradient system (mobile phase) is in 200 μ
L/min.Sample volume is 10 μ L.
3), Mass Spectrometer Method
The resulting eluent of step 2) is directly subjected to Mass Spectrometer Method.
Mass spectrometry experiments use QSTAR level four bars/time of-flight mass spectrometer, are equipped with TurboIonSpray ion source；Anion
Mode detection；Scanning range is 500~1000Da.Removing cluster voltage (DP) is 50V；Focusing potential (FP) is 200V；Ion source electricity
Pressure is 4500V；Removing cluster potential 2 (DP2) is 10V；Gas source 1 (GS1) is 50psi；Gas source 2 is (GS2) 45psi；Gas curtain gas (CUR)
For 40psi；Temperature is 400 DEG C；The impact energy that daughter ion scanning (PIS) uses is 20V.Data collection and analysis uses
Analyst QS v2.0。
As a result as follows:
One, the new prawn lipid-soluble extract of knife volume (that is, lipid crude extract of step 1)) is carried out by above-mentioned experimental method
HILIC-TOF/MS detection.The retention time that PE, PS and PC are detected according to eluting order is respectively 14.82,19.90 Hes
49.87 minutes (Fig. 1), wherein every one kind phosphatide includes multiple fatty acid chains phospholipid molecule different with saturation degree.Complicated
Phospholipid molecule ingredient produces certain influence, especially content PC the most abundant to the resolution ratio of chromatographic peak and peak shape.With mark
Quasi- product concentration range 0.5-200 μ g/mL carries out gradient dilution sample introduction, and standard items content is abscissa, and corresponding peak area is vertical
Coordinate, with y=a+bx linear fit, the standard curve for obtaining PE, PS and PC is y=1.1 × 107x+12658；Y=2.1 ×
107x+98919；With y=6.3 × 106- 29680, peak area (x) can be obtained according to by Fig. 1, PE, PS is calculated through standard curve
It is respectively as follows: 827,106 and 1138 μ g/g with the total content (y) of PC.
Identification is analyzed by mass spectrometry to each molecular species in PC, PE and PS three classes phosphatide, as a result such as Fig. 2.
PC is mainly with [M+HCOO]?Ion mode exists, and type is more, is concentrated mainly on 800 to 950 region m/z.Its
Maximum middle abundance is the ion of m/z 850.8 and 878.8, is respectively PC-16:0/22 through search lipidview database identification:
6&16:1/22:5 and PC-18:0/22:6&20:1/20:5.
PE molecule is then with [M-H]?Form exists, and responding maximum is m/z 786.6, its identified two fatty acid chains
Sn-1/sn-2 is 18:2/22:6.
PS ion is equally most strong with 786.6 signal of m/z, but structure is completely different, sn-1/sn-2 PS-18:0/
18:2.In addition it responds stronger also m/z 806.6 and is accredited as PS-16:0/22:6.And 23 PC molecules are identified, 18 PE
Molecule and 16 PS molecules amount to 57 phospholipid molecules (table 1).
The structure and content of PC, PE and PS phospholipid molecule kind in table 1, the new prawn of knife volume
The new prawn content of phospholipid of the new knife volume of knife volume is abundant, especially highest for the content of phospholipid of DHA or EPA chain with sn-2,
Such as 16:0/22:6,16:0/20:, 18:0/20:5.
Two, methodology validation
Gradient dilution sample introduction is carried out with 0.5~200 μ g/mL of standard concentration range, standard items content is abscissa, corresponding
Peak area be ordinate, with y=a+bx linear fit, the R of three kinds of models2It is all larger than 0.99.Respectively with 3 times and 10 times of noises
Than the detection limit (LOD) and quantitative limit (LOQ) that (S/N) determines target analytes, the detection for measuring PC, PE and PS is limited to 0.17~
0.28 μ g/mL is quantitatively limited to 0.54~0.80 μ g/mL.It is respectively LOQ and 20 times to blank muscle tissue sample pitch-based sphere
3 samples are arranged in LOQ, each concentration, and by method measurement is drafted, calculating is in a few days shown in Table 2 with day to day precision, the rate of recovery.In a few days
For precision less than 6.2%, day to day precision shows that precision is good less than 8.1%；The phosphatide rate of recovery is 74%~87%, full
Foot analysis test needs.
The parameters such as detection limit, accuracy, rate of recovery of phosphatide HILIC-TOF/MS detection method in table 2, the new prawn of knife volume
Methodology validation
In conclusion using hydrophilic chromatographic-tandem mass spectrum detection method of phosphatide in a kind of new prawn of knife volume of the present invention, altogether
7 molecules of PC, PE and PS three classes phosphatidase 5 are determined, the phosphatide in the new prawn of knife volume is rich in polyunsaturated fatty acid chain, sn-2
Position is especially in the majority with DHA chain and EPA chain.This method has quick convenient and sensitivity, precision and accuracy good, with tradition
Normal-phase chromatography, which is compared, avoids complicated flow visualizing and expensive normal-phase chromatography device, can be iipidomic in aquatic food
Certain basis has been established in development in subject.
Confirmatory experiment 1 divides the sample of embodiment 1 using the high positive chromatography of the generally acknowledged detection accuracy of the present invention
From quantitative, collection component progress Mass Spectrometer Method, acquired results are as follows: the total content (y) of PE, PS and PC are respectively as follows: 806,98 and
1105μg/g；Structural Identification result and table 1 are consistent.
Comparative example 1-1, by 1 step 2) of embodiment " acetonitrile solution of 53mmol/L formic acid be mobile phase A, 60mM formic acid
The aqueous solution of ammonium and 53mM formic acid is Mobile phase B " it is changed to that " acetonitrile solution of 26mmol/L formic acid is mobile phase A, 30mM ammonium formate
Aqueous solution with 26mM formic acid is Mobile phase B "；Remaining is equal to embodiment 1.Acquired results are the total content (y) of PE, PS and PC
It is respectively as follows: 613,58 and 705 μ g/g.
Partial primary phospholipid components content such as the following table 3, content are below content corresponding to the method for the present invention.
Table 3
Comparative example 1-2,
By 1 step 2) of embodiment " acetonitrile solution of 53mmol/L formic acid be mobile phase A, 60mM ammonium formate and 53mM first
The aqueous solution of acid is Mobile phase B " it is changed to " pure acetonitrile solution is mobile phase A, and pure water is Mobile phase B "；Remaining is equal to embodiment
1.Acquired results are that the total content (y) of PE, PS and PC are respectively as follows: 419,37 and 545 μ g/g.
Partial primary phospholipid components content such as the following table 4, content are below the content in the method for the present invention.
Table 4
Comparative example 2,
By the gradient system of 1 step 2) of embodiment by " 0~15min maintains 2% mobile phase A；15~60min will flow
Phase A is increased to 40% from 2%；60~70min maintains 40% mobile phase A；70~80min drops back to mobile phase A just from 40%
The 2% " of beginning state is changed to " the isocratic system of 20% Mobile phase B "；Remaining is equal to embodiment 1.Acquired results are PE's, PS and PC
Total content (y) is respectively as follows: 533,46 and 629 μ g/g.Structure of phospholipid qualification result is substantially the same as table 1.
Comparative example 3,
The flow velocity of 1 step 2) of embodiment is changed to " 400 μ L/min " by " 200 μ L/min "；Remaining is equal to embodiment 1.
Acquired results are that the total content (y) of PE, PS and PC are respectively as follows: 501,41 and 608 μ g/g.The substantially same table of structure of phospholipid qualification result
1。
Comparative example 4 " will go cluster voltage (DP) for 50V in 1 step 3) of embodiment；Temperature is 400 DEG C；Daughter ion scanning
(PIS) impact energy used is changed to that " removing cluster voltage (DP) is 40V for 20V "；Temperature is 400 DEG C；Daughter ion scans (PIS) and uses
Impact energy be 10V "；Remaining is equal to embodiment 1.Acquired results are that the total content (y) of PE, PS and PC are respectively as follows: 347,17
With 425 μ g/g.
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair
Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (6)
1. the hydrophilic chromatographic of phosphatide-tandem mass spectrum detection method in the new prawn of knife volume, it is characterised in that the following steps are included:
1), sample pre-treatments:
Knife volume is newly ground into shrimp gruel to shrimp, according to the solid-liquid ratio of 1g/15~20ml, after organic solvent I is added in shrimp gruel
Concussion mixes；Then it carries out ultrasonic ice bath to extract, pure water is added after extraction and shakes mixing, obtain mixture, every 1g shrimp is rotten
The ultrapure water of 10~15mL of adapted；Organic solvent I is the mixed liquor of methanol and chloroform, the volume ratio of methanol and chloroform is 0.5~
2:1；
Mixture is centrifuged with refrigerated centrifuge, pipettes lower phase solution from centrifugation gains；After the lower phase solution low temperature drying,
It is redissolved with organic solvent II, obtains lipid crude extract；The organic solvent II is the methanol aqueous solution or first of volumetric concentration >=30%
Alcohol；
2), liquid phase separation:
Lipid crude extract is subjected to liquid phase separation；Chromatographic column is YMC Triart glycol-based HILIC column；Chromatographic condition uses gradient
Elution method, gradient system are made of mobile phase A and Mobile phase B；Wherein mobile phase A is that the acetonitrile containing 53mmol/L formic acid is molten
Liquid, Mobile phase B are the aqueous solution containing 60mM ammonium formate and 53mM formic acid；
Gradient system are as follows: 0~15min maintains 2% mobile phase A；Mobile phase A is increased to 40% from 2% by 15~60min；60
~70min maintains 40% mobile phase A；Mobile phase A is dropped back to the 2% of original state from 40% by 70~80min；Above-mentioned % is
Volume %；
Flow velocity when gradient elution is 180~220 μ L/min；
3) the resulting eluent of step 2), is subjected to Mass Spectrometer Method analysis；
Using QSTAR level four bars/time of-flight mass spectrometer, it is equipped with TurboIonSpray ion source；Negative ion mode detection；It sweeps
Retouching range is 500~1000Da；Removing cluster voltage is 50V；Focusing potential is 200V；Ion source voltage is 4500V；Remove cluster potential 2
For 10V；Gas source 1 is 50psi；Gas source 2 is 45psi；Gas curtain gas is 40psi；Temperature is 400 DEG C；What daughter ion scanning used touches
Hitting can be 20V；Data collection and analysis uses Analyst QS v2.0.
2. hydrophilic chromatographic-tandem mass spectrum detection method of phosphatide, feature exist in the new prawn of knife volume according to claim 1
In:
It is rotten to pipette the substitution shrimp of the centrifugation gains after lower phase solution in the step 1), with the chloroform substitution in organic solvent I
Organic solvent I is repeated and is extracted 1~3 time；
Merge after the resulting lower phase solution of all extraction steps merges and carries out subsequent drying and redissolution.
3. hydrophilic chromatographic-tandem mass spectrum detection method of phosphatide in the new prawn of knife volume according to claim 1 or 2, special
Sign is:
Ultrasound condition is 25kHz in the step 1).
4. hydrophilic chromatographic-tandem mass spectrum detection method of phosphatide in the new prawn of knife volume according to claim 1 or 2, special
Sign is:
Refrigerated centrifuge is in 4 DEG C with 8000rpm centrifugation 15 minutes in the step 1).
5. hydrophilic chromatographic-tandem mass spectrum detection method of phosphatide in the new prawn of knife volume according to claim 1 or 2, special
Sign is:
Sample volume is 5~10 μ L in the step 2).
6. hydrophilic chromatographic-tandem mass spectrum detection method of phosphatide in the new prawn of knife volume according to claim 1 or 2, special
Sign is:
In the step 1), the solid-liquid ratio of the organic solvent II of shrimp gruel and redissolution is 1g/1~2ml.
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