CN106153763A - The hydrophilic chromatographic tandem mass spectrum detection method of phospholipid in the new prawn of cutter volume - Google Patents
The hydrophilic chromatographic tandem mass spectrum detection method of phospholipid in the new prawn of cutter volume Download PDFInfo
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Abstract
The invention discloses the hydrophilic chromatographic tandem mass spectrum detection method of phospholipid in a kind of new prawn of cutter volume, comprise the following steps: 1), sample pre-treatments: carry out corresponding pre-treatment after cutter volume is newly ground to form shrimp gruel to Macrobrachium nipponensis, obtain lipid crude extract；2), liquid phase separation: lipid crude extract is carried out liquid phase separation；Chromatographic column is YMC Triart glycol-based HILIC post；Chromatographic condition uses linear gradient elution method, and gradient system is made up 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 containing 60mM ammonium formate and 53mM first aqueous acid；3), by step 2) eluent of gained carries out Mass Spectrometer Method analysis.The method using the present invention can accurately detect the total content of PE, PS and PC in sample, and realize to PC, PE and PS tri-in class phospholipid each molecular species carry out mass spectral analysis qualification.
Description
Technical field
The invention belongs to field of food detection, relate to the hydrophilic chromatographic of phospholipid in a kind of new prawn of cutter volume-tandem mass spectrum inspection
Survey method, refers specifically to phosphatidylcholine in a kind of new prawn of cutter volume, PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine three major types phospholipid
Structural Identification and quantitative analysis method.
Background technology
The new prawn of cutter volume (Metapenaeus ensis), i.e. base encloses shrimp, is that China cultivates one of small-sized marine products shrimps.Cause
It has the advantages such as feeding habits are miscellaneous, it is fast, high temperature resistant to grow, proper salinity level is wide, anti-poor environment ability is strong, in recent years Central-South coastal
Artificial cultivation has extensively been carried out in area, demonstrates good economic outlook.Shrimps aquatic resources lipid content is high, rich in Ω-3 race
With Ω-6 race's polyunsaturated fatty acid and phosphatidylcholine (Phosphatidylcholine, PC), PHOSPHATIDYL ETHANOLAMINE
The phospholipid substance such as (Phosphatidylethanolamine, PE), Phosphatidylserine (Phosphatidlserine, PS).
Phospholipid is the lipid that a class contains phosphoric acid.Structure of phospholipid mainly by glycerol backbone, polar group and different length and
The fatty acid chain composition of saturation.Different according to phospholipid polar group, phospholipid can be divided into phosphatidylcholine, phosphatidyl ethanol
The classification such as amine, Phosphatidylserine.Present in organism, phospholipid kind is thousands of, and various structures, kind are complicated, have uniqueness
Chemical constitution.Phospholipid is to maintain cyto-architectural important component part, is also the indispensable nutrient of metabolism.Phosphorus
Fat is also equipped with biological function widely, such as activating cell, and maintenance metabolism is Jun Heng with hormone secretion, strengthens body immunity
Deng.It is demonstrated experimentally that phospholipid can reduce blood lipid, cholesterol, stop superabundant fats to deposit in blood vessel wall, make vascular circulation smooth and easy,
It is described as " blood vessel street cleaner ".In the efficient new prawn of cutter volume, the detection method of phospholipid molecule is beneficial to deepen the new prawn of cutter volume
The Nutritional studies of middle phospholipid, promotes that the new prawn of cutter volume is the product development of raw material.
Tradition phospholipid detection method includes high performance liquid chromatography (HPLC), NMR (Nuclear Magnetic Resonance) spectrum (NMR), fluorescence spectrum
(FLR) etc., these methods analysts time-consumingly long, sensitivity is low.Hydrophilic chromatographic (HILIC) is to separate highly polar and hydrophilic specially
A kind of method of compound.Because using conventional reversed phase chromatography flowing phase, hydrophilic chromatographic is made to have preferable mass spectrum compatible.
Current existing lipid separation conventional method is normal-phase chromatography, needs special chromatographic equipment (just own with flowing phase
Alkane, isopropanol, chloroform), and the eluent of this flowing phase gained directly cannot be 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 and simple to handle, new prawn of cutter volume that qualitative, quantitative ability is stronger
The hydrophilic chromatographic of middle phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE and Phosphatidylserine-tandem mass spectrum detection method.
In order to solve above-mentioned technical problem, the present invention provides the hydrophilic chromatographic-series connection matter of phospholipid in a kind of new prawn of cutter volume
Spectrum detection method, comprises the following steps:
1), sample pre-treatments:
Cutter volume is newly ground to form shrimp rotten to Macrobrachium nipponensis, according to the solid-liquid ratio of 1g/15～20ml, shrimp gruel adds organic solvent
Concussion mixing after I；Then carry out ultrasonic (probe type ultrasonic) ice bath and extract (extraction time is 10～15 minutes), after extraction
Add pure water and shake mixing, obtaining mixture, the ultra-pure water of every 1g shrimp gruel adapted 10～15mL；Organic solvent I is methanol and chlorine
Imitative mixed liquor, methanol is 0.5～2:1 with the volume ratio of chloroform；
Mixture refrigerated centrifuger is centrifuged, and pipettes lower phase solution from centrifugal gains；Described lower phase solution low temperature (≤
10 DEG C) it is dried after (carrying out low temperature with nitrogen to dry up), redissolve with organic solvent II, obtain lipid crude extract；Described organic solvent II
It is methanol aqueous solution or methanol (that is, 30～the methanol aqueous solution of 100%, the methanol of preferably 90% of volumetric concentration >=30%
Aqueous solution),
2), liquid phase separation:
Lipid crude extract is carried out liquid phase separation；Chromatographic column be YMC Triart glycol-based HILIC post (4.6 × 250mm, 3
μm).Chromatographic condition uses linear gradient elution method, and gradient system is made up of mobile phase A and Mobile phase B；Wherein mobile phase A is for containing
The acetonitrile solution (pH is about 4.0～4.5) of 53mmol/L formic acid, Mobile phase B is containing 60mM ammonium formate and the water of 53mM formic acid
Solution (pH is about 3.6)；
Gradient system is: 0～15min, maintains 2% mobile phase A；15～60min, mobile phase A is brought up to from 2%
40%；60～70min, maintain 40% mobile phase A；70～80min, mobile phase A is dropped back to from 40% the 2% of original state；On
State % and be volume %；
Remarks illustrate: the eluent of liquid phase is directly entered mass spectral analysis；
Liquid chromatograph uses Agilent 1100 system, is equipped with the units such as quaternary pump, automatic sampler, chromatographic column calorstat
Part；
3), by step 2) eluent of gained carries out Mass Spectrometer Method analysis.
As the improvement of the hydrophilic chromatographic of phospholipid-tandem mass spectrum detection method in the new prawn of cutter volume of the present invention:
Described step 3):
Use QSTAR level Four bar/time of-flight mass spectrometer, be equipped with TurboIonSpray ion source；Negative ion mode is examined
Survey；Sweep limits is 500～1000Da；Removing a bunch voltage (DP) is 50V；Focusing potential (FP) is 200V；Ion source voltage is
4500V；Removing bunch electromotive force 2 (DP2) is 10V；Source of the gas 1 (GS1) is 50psi；Source of the gas 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 illustrate: phospholipid can be separated by liquid chromatograph by class, and such as Fig. 1, each peak represents a class phospholipid, and (some peak is miscellaneous
Matter), in every class phospholipid, different different from double bond quantity according to carbon chain lengths, comprise a lot of phospholipid molecule, mass spectrum is to every class phospholipid
After detection, such as Fig. 2, (i.e. the ratio of quality and electric charge, in this experiment can to obtain the mass-to-charge ratio of the different phospholipid molecules of every class phospholipid
Electric 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 phospholipid
Amount, then search the most available structural information in storehouse through lipidview.
As the further improvement of the hydrophilic chromatographic of phospholipid-tandem mass spectrum detection method in the new prawn of cutter volume of the present invention:
Described step 1) in, (with the centrifugal gains after pipetting lower phase solution that is, include upper phase solution, be positioned at and mix
Solids between liquid and lower phase solution) substitute shrimp gruel, substitute organic solvent I with the chloroform in organic solvent I, repeat and carry out
Extract 1～3 time；
Follow-up being dried and redissolve is carried out after merging the lower phase solution merging of all extraction step gained.
Remarks illustrate: lower leaf on solution in centrifuge tube after centrifugal, cutter volume newly shrimp meat is then occupy in pie biphase in
Between.
Methanol, chloroform, water three together when will not be miscible, can be divided into the most biphase, methanol polarity more by force can be with
Water is miscible, and therefore in centrifuge tube, the miscible a small amount of methanol of chloroform is in lower phase, and water miscible volume methanol is in upper phase；Owing to pipetting away
After lower phase, actual major part methanol is not still pipetted in upper phase, therefore repeats to have only to the when of extraction add chloroform
?.That is, the volumetric usage of the chloroform in the volumetric usage=machine solvent I of chloroform when repeating to extract.
As the further improvement of the hydrophilic chromatographic of phospholipid-tandem mass spectrum detection method in the new prawn of cutter volume of the present invention:
Described step 1) in ultrasound condition be 25kHz.
As the further improvement of the hydrophilic chromatographic of phospholipid-tandem mass spectrum detection method in the new prawn of cutter volume of the present invention:
Described step 1) in frozen centrifugation for be centrifuged 15 minutes in 4 DEG C with 8000rpm.
As the further improvement of the hydrophilic chromatographic of phospholipid-tandem mass spectrum detection method in the new prawn of cutter volume of the present invention:
Described step 2) in, flow velocity during gradient elution is 180～220 μ L/min (preferably 200 μ L/min).
As the further improvement of the hydrophilic chromatographic of phospholipid-tandem mass spectrum detection method in the new prawn of cutter volume of the present invention:
Described step 2) in sample size (that is, the amount of lipid crude extract) be 5～10 μ L.
As the further improvement of the hydrophilic chromatographic of phospholipid-tandem mass spectrum detection method in the new prawn of cutter volume of the present invention:
Described step 1) in, rotten and the organic solvent II redissolved the solid-liquid ratio of shrimp is 1g/1～2ml.
The step 1 of the present invention) sample pre-treatments in:
Take the cutter volume new prawn muscle tissue sample after homogenizing (shrimp is rotten) concussion after centrifuge tube, addition organic solvent I mixed
Even.Probe type ultrasonic ice bath extracts, and complete backward centrifuge tube adds pure water and shakes mixing.Mixture refrigerated centrifuger from
The heart, lower leaf on solution in centrifugal rear centrifuge tube, cutter volume new prawn muscle tissue sample then occupy biphase centre in pie.With shifting
Lower phase solution is transferred to another new pipet by liquid rifle, continues up mutually and adds chloroform in sample and repeat aforesaid operations
Extract 1～3 time (for example, twice).The organic facies extracted is merged, dries up with nitrogen low temperature, and redissolve with methanol aqueous solution.
In the present invention, cross the PTFE filter membrane of 0.22 μm after redissolution, obtain lipid crude extract.
The present invention uses glycol-based HILIC post, with the acetonitrile solution of 53mmol/L formic acid as mobile phase A, and 60mM ammonium formate
It is Mobile phase B with 53mM first aqueous acid；After gradient elution (0～15min, maintain 2% mobile phase A；15～60min, will
Mobile phase A brings up to 40% from 2%；60～70min, maintain 40% mobile phase A；70～80min, by mobile phase A from 40% fall
Return the 2% of original state) phospholipid is separated.Comparing conventional chromatogram technology, the method can make lipid separate at laboratory routine instrument
In device reversed phase chromatography accomplished, effectively prevent the equipment needed for tradition normal-phase chromatography and flowing phase, use methanol or acetonitrile
, and directly can be combined with mass spectrum.
Prepared by the detection method sample of the present invention, liquid phase separation, Mass Spectrometer Method, easily training easy and simple to handle, simple, qualitative fixed
Amount ability is stronger, practical, and applicable laboratory is widely used.
In sum, the hydrophilic chromatographic-tandem mass spectrum detection method of phospholipid in a kind of new prawn of cutter volume that the present invention sets up
Simple and quick, highly sensitive, result is reliable and stable, significant for the development of aquatic products iipidomic.Use this
The method of invention can accurately detect the total content of PE, PS and PC in sample, and realize to PC, PE and PS tri-in class phospholipid each
Individual molecular species carries out mass spectral analysis qualification.
Accompanying drawing explanation
Below in conjunction with the accompanying drawings the detailed description of the invention of the present invention is described in further detail.
Fig. 1 is the HILIC-TOF/MS detection figure of cutter volume new prawn muscular tissue lipid-soluble extract；
TIC of+TOF MS:from Sample of 20150610.wiff (Turbo Spray).
The mass spectrum of PC, PE and PS in Fig. 2 position cutter volume new prawn lipid-soluble extract；
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)
Figure below (PS):
+ TOFMS:48.899to49.449min from Sample7of 20150610.wiff
A=3.59917355969297020e-004, t0=-5.28098098505142840e+000 (Turbo
Spray)。
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.
In the present invention:
Material and reagent
Three kinds of phospholipid standard substance: PC-14:0/14:0, PE-15:0/15:0 and PS-14:0/14:0 purity is all higher than
99.9% (U.S. Avanti Polar Lipids company)；The standard substance mother solution of 1mg/mL it is configured to methanol for solvent.Chlorine
The reagent such as imitative, methanol, acetonitrile are bought from Merck company of Germany.Resistivity is that the ultra-pure water of 18.2M Ω cm filters from Milli-
Q system (Millipore company of the U.S.).
Hydrophilic chromatographic-tandem mass spectrum the detection method of phospholipid in embodiment 1, a kind of new prawn of cutter volume
It is specially and follows the steps below successively:
1), sample pre-treatments:
After the new prawn of cutter volume removes excretory gland, take abdominal part striped muscle meat tissue and as Macrobrachium nipponensis and grind to form the rotten (slurry of shrimp
Shape).Weigh 1.0g shrimp rotten in 5mL centrifuge tube, add 18mL chloroform/methanol mixed liquor (1:2, v/v) and shake mixing afterwards.At 250W
Lower ultrasound wave (25kHz) ice bath extracts 10 minutes backward centrifuge tubes and adds 12.5mL ultra-pure water and shake mixing.Mixture is with cold
Freeze centrifuge (Thermo Scientific company of the U.S.) to be centrifuged 5 minutes with 8000g in 4 DEG C, in centrifugal rear centrifuge tube on solution
Lower leaf, tissue sample then occupy biphase centre in pie.With liquid-transfering gun, lower phase solution is transferred to another new pipet,
Continue up mutually and sample adds 6mL chloroform and repeat aforesaid operations extraction twice.By three times extract organic facies (under mix
Liquid) merge, dry up with nitrogen low temperature, and cross 0.22 μm after methanol aqueous solution 2ml (methanol: water=9:1, v/v) redissolution
PTFE filter membrane.
2), liquid phase separation
Liquid chromatograph experiment uses Agilent 1100 system, is equipped with quaternary pump, automatic sampler, chromatographic column calorstat etc.
Element.Chromatographic column is YMC Triart glycol-based HILIC post (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 53mM first aqueous acid (pH is about 3.6).
Gradient system is made up of mobile phase A and Mobile phase B；Gradient system is as follows: 0～15min, maintains 2% mobile phase A；
15～60min, mobile phase A is brought up to 40% from 2%；60～70min, maintain 40% mobile phase A；70～80min, will flowing
Phase A drops back to original state from 40%, and i.e. 2%.Above-mentioned % is volume %.The flow speed control of gradient system (flowing phase) is at 200 μ
L/min.Sample size is 10 μ L.
3), Mass Spectrometer Method
By step 2) eluent of gained directly carries out Mass Spectrometer Method.
Mass spectrometry experiments uses QSTAR level Four bar/time of-flight mass spectrometer, is equipped with TurboIonSpray ion source；Anion
Mode detection；Sweep limits is 500～1000Da.Removing a bunch voltage (DP) is 50V；Focusing potential (FP) is 200V；Ion source electricity
Pressure is 4500V；Removing bunch electromotive force 2 (DP2) is 10V；Source of the gas 1 (GS1) is 50psi；Source of the gas 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。
Result is as follows:
One, by the lipid crude extract of above-mentioned experimental technique prawn new to cutter volume lipid-soluble extract (that is, step 1)) carry out
HILIC-TOF/MS detects.Detect that according to eluting order the retention time of PE, PS and PC is respectively 14.82,19.90 Hes
Minute 49.87 (Fig. 1), each of which class phospholipid all comprises the phospholipid molecule that multiple fatty acid chain is different with saturation.Complicated
Phospholipid molecule composition creates certain impact, the PC that especially content enriches the most on the resolution of chromatographic peak with peak shape.With mark
Quasi-product concentration range 0.5-200 μ g/mL carries out gradient dilution sample introduction, and standard substance content is abscissa, and corresponding peak area is vertical
Coordinate, with y=a+bx linear fit, the standard curve 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, calculate PE, PS through standard curve
It is respectively as follows: 827,106 and 1138 μ g/g with the total content (y) of PC.
To PC, PE and PS tri-in class phospholipid each molecular species carry out mass spectral analysis qualification, result such as Fig. 2.
PC is mainly with [M+HCOO]-Ion mode exists, and kind is more, is concentrated mainly on m/z 800 to 950 region.Its
Middle abundance maximum for m/z 850.8 and 878.8 ion, identify through search lipidview data base and be respectively PC-16:0/22:
6&16:1/22:5 and PC-18:0/22:6&20:1/20:5.
PE molecule is then with [M-H]-Form exists, response maximum for m/z 786.6, its two fatty acid chains identified
Sn-1/sn-2 is 18:2/22:6.
PS ion is the strongest with m/z 786.6 signal equally, but structure is completely different, and its sn-1/sn-2 is PS-18:0/
18:2.Additionally respond the stronger m/z 806.6 that also has and be accredited as PS-16:0/22:6.And identify 23 PC molecules, 18 PE
Molecule and 16 PS molecules, altogether 57 phospholipid molecules (table 1).
The structure of PC, PE and PS phospholipid molecule kind and content in table 1, the new prawn of cutter volume
Cutter volume new cutter volume new prawn content of phospholipid enriches, especially with sn-2 as DHA or the content of phospholipid of EPA chain is the highest,
Such as 16:0/22:6,16:0/20:, 18:0/20:5 etc..
Two, Method validation
Carrying out gradient dilution sample introduction with standard concentration scope 0.5～200 μ g/mL, standard substance content is abscissa, corresponding
Peak area be vertical coordinate, with y=a+bx linear fit, the R of three kinds of models2It is all higher than 0.99.Respectively with 3 times and 10 times of noises
Determine detection limit (LOD) and the quantitative limit (LOQ) of target analytes than (S/N), record the detection of PC, PE and PS be limited to 0.17～
0.28 μ g/mL, is quantitatively limited to 0.54～0.80 μ g/mL.Blank muscle tissue sample pitch-based sphere is respectively LOQ and 20 times
LOQ, each concentration arranges 3 samples, measures by drafting method, calculates and be in a few days shown in Table 2 with day to day precision, the response rate.In a few days
Precision is less than 6.2%, and day to day precision is less than 8.1%, shows that precision is good；The phospholipid response rate is 74%～87%, full
Foot analyzes test to be needed.
The parameter such as the detection limit of phospholipid HILIC-TOF/MS detection method, accuracy, response rate in table 2, the new prawn of cutter volume
Method validation
In sum, utilize the hydrophilic chromatographic-tandem mass spectrum detection method of phospholipid in the present invention a kind of cutter new prawn of volume, altogether
Determine PC, PE and PS 7 molecules of tri-class phosphatidase 5s, the phospholipid in the new prawn of cutter volume rich in polyunsaturated fatty acid chain, its sn-2
Position is especially in the majority with DHA chain and EPA chain.The method has the most convenient and sensitivity, precision and accuracy are good, with tradition
Normal-phase chromatography is compared and is avoided the flow visualizing of complexity and expensive normal-phase chromatography device, can be that iipidomic is at aquatic food
Certain basis has been established in development in subject.
Confirmatory experiment 1, the forward chromatography that the accuracy of detection generally acknowledged of the present invention is high is used the sample of embodiment 1 to be carried out point
From quantitatively, collecting component and carry out Mass Spectrometer Method, acquired results is: the total content (y) of PE, PS and PC is respectively as follows: 806,98 and
1105μg/g；Structural Identification result is consistent with table 1.
Comparative example 1-1, by embodiment 1 step 2) " acetonitrile solution of 53mmol/L formic acid is mobile phase A, 60mM formic acid
Ammonium and 53mM first aqueous acid are Mobile phase B " make that " acetonitrile solution of 26mmol/L formic acid is mobile phase A, 30mM ammonium formate into
It is Mobile phase B with 26mM first aqueous acid "；Remaining is equal to embodiment 1.Acquired results is the total content (y) of PE, PS and PC
It is respectively as follows: 613,58 and 705 μ g/g.
The primary phospholipid components content such as table 3 below of part, its content is below the content corresponding to the inventive method.
Table 3
Comparative example 1-2,
By embodiment 1 step 2) " acetonitrile solution of 53mmol/L formic acid is mobile phase A, 60mM ammonium formate and 53mM first
Aqueous acid is Mobile phase B " make " pure acetonitrile solution is mobile phase A, and pure water is Mobile phase B " into；Remaining is equal to embodiment
1.Acquired results is that the total content (y) of PE, PS and PC is respectively as follows: 419,37 and 545 μ g/g.
The primary phospholipid components content such as table 4 below of part, content is below the content in the inventive method.
Table 4
Comparative example 2,
By embodiment 1 step 2) gradient system by " 0～15min, maintain 2% mobile phase A；15～60min, will flowing
Phase A brings up to 40% from 2%；60～70min, maintain 40% mobile phase A；70～80min, by mobile phase A from 40% drop back at the beginning of
The 2% of beginning state " make " the 20% isocratic system of Mobile phase B " into；Remaining is equal to embodiment 1.Acquired results is PE, PS and PC's
Total content (y) is respectively as follows: 533,46 and 629 μ g/g.Structure of phospholipid qualification result is substantially with table 1.
Comparative example 3,
By embodiment 1 step 2) flow velocity made into " 400 μ L/min " by " 200 μ L/min "；Remaining is equal to embodiment 1.
Acquired results is that the total content (y) of PE, PS and PC is respectively as follows: 501,41 and 608 μ g/g.The most same table of structure of phospholipid qualification result
1。
Comparative example 4, by embodiment 1 step 3) in " removing a bunch voltage (DP) is 50V；Temperature is 400 DEG C；Daughter ion scans
(PIS) impact energy used is 20V " make that " removing a bunch voltage (DP) is 40V into；Temperature is 400 DEG C；Daughter ion scanning (PIS) uses
Impact energy be 10V "；Remaining is equal to embodiment 1.Acquired results is that the total content (y) of PE, PS and PC is respectively as follows: 347,17
With 425 μ g/g.
Finally, in addition it is also necessary to be only several specific embodiments of the present invention it is noted that listed above.Obviously, this
Bright it is not limited to above example, it is also possible to have many deformation.Those of ordinary skill in the art can be from present disclosure
The all deformation directly derived or associate, are all considered as protection scope of the present invention.
Claims (8)
1. hydrophilic chromatographic-tandem mass spectrum the detection method of phospholipid in the new prawn of cutter volume, it is characterised in that comprise the following steps:
1), sample pre-treatments:
Cutter volume is newly ground to form shrimp rotten to Macrobrachium nipponensis, according to the solid-liquid ratio of 1g/15～20ml, after adding organic solvent I in shrimp gruel
Concussion mixing；Then carrying out ultrasonic ice bath extraction, add pure water and shake mixing, obtain mixture after extraction, every 1g shrimp is rotten
The ultra-pure water of adapted 10～15mL；Organic solvent I is the mixed liquor of methanol and chloroform, the volume ratio of methanol and chloroform be 0.5～
2:1；
Mixture refrigerated centrifuger is centrifuged, and pipettes lower phase solution from centrifugal gains；After described lower phase solution cold drying,
Redissolve with organic solvent II, obtain lipid crude extract；Described organic solvent II is methanol aqueous solution or the first of volumetric concentration >=30%
Alcohol,
2), liquid phase separation:
Lipid crude extract is carried out liquid phase separation；Chromatographic column is YMC Triart glycol-based HILIC post；Chromatographic condition uses gradient
Elution method, gradient system is made up 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 is containing 60mM ammonium formate and 53mM first aqueous acid；
Gradient system is: 0～15min, maintains 2% mobile phase A；15～60min, mobile phase A is brought up to 40% from 2%；60
～70min, maintain 40% mobile phase A；70～80min, mobile phase A is dropped back to from 40% the 2% of original state；Above-mentioned % is
Volume %；
3), by step 2) eluent of gained carries out Mass Spectrometer Method analysis.
Hydrophilic chromatographic-tandem mass spectrum the detection method of phospholipid in the new prawn of cutter volume the most according to claim 1, its feature exists
In:
Described step 3):
Use QSTAR level Four bar/time of-flight mass spectrometer, be equipped with TurboIonSpray ion source；Negative ion mode detects；Sweep
The scope of retouching is 500～1000Da；Removing bunch voltage is 50V；Focusing potential is 200V；Ion source voltage is 4500V；Remove bunch electromotive force 2
For 10V；Source of the gas 1 is 50psi；Source of the gas 2 is 45psi；Gas curtain gas is 40psi；Temperature is 400 DEG C；What daughter ion scanning used touches
Hitting can be for 20V；Data collection and analysis uses Analyst QS v2.0.
Hydrophilic chromatographic-tandem mass spectrum the detection method of phospholipid in the new prawn of cutter volume the most according to claim 1, its feature exists
In:
Described step 1) in, substitute shrimp with the centrifugal gains after pipetting lower phase solution rotten, substitute with the chloroform in organic solvent I
Organic solvent I, repeats and carries out extracting 1～3 time；
Follow-up being dried and redissolve is carried out after merging the lower phase solution merging of all extraction step gained.
4. according to the hydrophilic chromatographic-tandem mass spectrum detection method of phospholipid in the arbitrary described new prawn of cutter volume of claims 1 to 3,
It is characterized in that:
Described step 1) in ultrasound condition be 25kHz.
5. according to the hydrophilic chromatographic-tandem mass spectrum detection method of phospholipid in the arbitrary described new prawn of cutter volume of claims 1 to 3,
It is characterized in that:
Described step 1) in frozen centrifugation for be centrifuged 15 minutes in 4 DEG C with 8000rpm.
6. according to the hydrophilic chromatographic-tandem mass spectrum detection method of phospholipid in the arbitrary described new prawn of cutter volume of claims 1 to 3,
It is characterized in that:
Described step 2) in, flow velocity during gradient elution is 180～220 μ L/min.
Hydrophilic chromatographic-tandem mass spectrum the detection method of phospholipid in the new prawn of cutter volume the most according to claim 6, its feature exists
In:
Described step 2) in sample size be 5～10 μ L.
8. according to the hydrophilic chromatographic-tandem mass spectrum detection method of phospholipid in the arbitrary described new prawn of cutter volume of claims 1 to 3,
It is characterized in that:
Described step 1) in, rotten and the organic solvent II redissolved the solid-liquid ratio of shrimp is 1g/1～2ml.
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