CN111812248A - Analysis and detection method for effectively screening phospholipids in krill oil - Google Patents
Analysis and detection method for effectively screening phospholipids in krill oil Download PDFInfo
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Abstract
The invention discloses an analysis and detection method for effectively screening phospholipids in krill oil, which comprises the following steps: grinding krill powder, adding into chloroform-methanol mixed solution, and oscillating at room temperature to obtain extractive solution A; carrying out ultrasonic treatment on the extract A, centrifuging the mixture, and collecting a lower organic phase and a centrifuged residue to obtain an organic phase B and a residue C; re-extracting the residue C with chloroform for several times, and collecting the lower organic phase to obtain organic phase D; mixing the organic phase B and the organic phase D to obtain an E1 extract, blow-drying the E1 extract, fixing the volume with acetonitrile, and filtering with an organic filter membrane to obtain a solution to be detected E; and finally, detecting the solution to be detected E by a hydrophilic interaction liquid chromatography-mass spectrometry combination method in a precursor ion scanning mode. The method can realize non-targeted screening of phospholipid in krill oil, and has the characteristics of high detection efficiency, high detection accuracy and high resolution.
Description
Technical Field
The invention relates to a detection method of krill oil, in particular to an analysis detection method for effectively screening phospholipids in krill oil.
Background
Antarctic krill is one of the most abundant organisms found in Antarctic waters of the south Ice ocean, and has great potential and application prospect, and krill oil in the Antarctic krill is often used as a substitute of fish oil on the market at present. Krill oil is rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which differ from omega-3 fatty acids of other biological origin in that krill oil contains high levels of omega-3 fatty acyl chains at the sn-2 position of phospholipids, whereas the omega-3 fatty acids contained in fish oil are usually in the form of triglycerides or ethyl esters. Because EPA type structure phospholipid and DHA type structure phospholipid can be easily combined to cell membranes and absorbed by intestinal tracts, the effects of reducing cardiovascular risks, improving chronic inflammation, improving brain function, treating premenstrual syndrome and the like are mentioned, krill oil containing the phospholipid is considered as a potential lipid source with important health care value, and how to accurately detect the EPA type structure phospholipid and the DHA type structure phospholipid in the krill oil becomes a prerequisite for realizing effective utilization of the phospholipid.
The traditional detection methods for phospholipids are divided into a shotgun method and a rapid evaporation ionization mass spectrometry method, but the two methods do not relate to chromatographic separation and are easily interfered by matrix effect and ion inhibition, so that the analysis result is poor. Under the promotion of mass spectrometry technology, lipidomics for carrying out systematic analysis on the whole lipid is rapidly developed, so that a liquid chromatography-mass spectrometry principle for realizing phospholipid detection by using lipidomics becomes a novel detection method for improving phospholipid detection efficiency and comprehensiveness. However, as the omics data is more complex, the method is easily interfered by the phospholipid with a saturated or short-chain structure in the detection process, so that the method has certain difficulty in the non-targeted screening of the EPA-type structure phospholipid and the DHA-type structure phospholipid with variable molecular weights.
Therefore, an effective method for non-targeted screening of EPA-type structural phospholipids and DHA-type structural phospholipids in antarctic krill oil is needed.
Disclosure of Invention
The invention aims to provide an analysis and detection method for effectively screening phospholipids in krill oil. The method can realize non-targeted screening of phospholipid in krill oil, and has the characteristics of high detection efficiency, high detection accuracy and high resolution.
The technical scheme of the invention is as follows: an analytical detection method for effectively screening phospholipids in krill oil comprises the following steps:
taking 10g of krill powder, grinding uniformly, adding 100-300 mL of chloroform-methanol mixed solution, and oscillating for 1-3 minutes at room temperature to obtain an extracting solution A;
secondly, performing ultrasonic treatment on the A extracting solution to release krill oil from a solid mechanism to obtain a B1 mixture, centrifuging the B1 mixture, and collecting a lower organic phase and a centrifuged residue to obtain a B organic phase and a C residue;
thirdly, re-extracting the C residues for multiple times by using 100-300 mL of trichloromethane, and collecting a lower organic phase to obtain a D organic phase;
mixing the organic phase B and the organic phase D to obtain an E1 extract, blow-drying the E1 extract, fixing the volume with acetonitrile, and filtering with an organic filter membrane to obtain a solution to be detected E;
and fifthly, detecting the solution to be detected E by a hydrophilic interaction liquid chromatography-mass spectrometry combined method in a precursor ion scanning mode.
In the analysis and detection method for effectively screening phospholipids in krill oil, the specific steps of the second step are as follows: and (3) performing ultrasonic-assisted extraction on the A extracting solution for 10-20 min under the conditions of 20kHz and 130W to obtain a B1 mixture, and then centrifuging the B1 mixture at 3-5 ℃ for 8-12 min by 8000g to obtain a B organic phase and a C residue. .
In the aforementioned analytical detection method for effectively screening phospholipids in krill oil, the specific step of the step (iv) is: and mixing the organic phase B and the organic phase D to obtain an E1 extract, blowing the E1 extract at the ambient temperature of 4 ℃ by using nitrogen flow, then fixing the volume by using 1mL of acetonitrile, and passing through a 0.22 mu m organic filter membrane to obtain a solution to be detected, wherein the solution to be detected is E.
In the aforementioned method for effectively screening phospholipids in krill oil, the liquid chromatography conditions of the liquid chromatography-mass spectrometry method in the fifth step are as follows: the liquid chromatographic column is a Cosmosil-HILIC chromatographic column; the sample introduction amount is 2 mu L, and the temperature of the chromatographic column is kept at 30 ℃; the mobile phase A is obtained by adding pure water into a mixed solution of 20mmol of ammonium formate and 20mmol of formic acid and fixing the volume to 1L; the mobile phase B is a formic acid acetonitrile solution with the concentration of 20mmol of the prepared formic acid.
In the aforementioned method for effectively screening phospholipids in krill oil, the liquid chromatography column elution method of the liquid chromatography-mass spectrometry method in the fifth step specifically comprises: when the time is 0-10 min, the content of the mobile phase B is uniformly reduced from 95% to 70%, when the time is 10-15 min, the content of the mobile phase B is uniformly reduced from 70% to 50%, and when the time is 15-20 min, the content of the mobile phase B is kept at 50%.
In the aforementioned method for effectively screening phospholipids in krill oil, the mass spectrometry mode of the liquid chromatography-mass spectrometry method in the fifth step is specifically as follows: injecting the solution to be tested into an electrospray ionization source by the sample injection amount of 2 mu L, scanning precursor ions in a negative ion mode, and setting product ions of EPA-type structure phospholipid and DHA-type structure phospholipid as m/z301 and m/z327 respectively.
In the aforementioned method for effectively screening phospholipids in krill oil, the mass spectrometry conditions of the liquid chromatography-mass spectrometry method in the fifth step are specifically as follows: setting the ion spray voltage at 4500V in the negative ion mode, the ion source temperature was maintained at 500 ℃; the gas curtain gas, the drying gas and the atomizer gas in the ion source gas are respectively set to be 35psi, 50psi and 60 psi; the mass spectrum measurement range is m/z 450-950, and the scanning speed is 1s per spectrum; data acquisition, processing and analysis are then completed.
In the analysis and detection method for effectively screening phospholipids in krill oil, in the fifth step, when the liquid chromatography-mass spectrometry is used for detecting phosphatidylcholine ions in the solution to be detected, the collision voltage is 50eV and the declustering voltage is 100eV under the mass spectrometry condition; when the liquid chromatography-mass spectrometry is used for detecting phosphatidylethanolamine ions in a solution to be detected, the collision voltage in the mass spectrometry condition is 40eV, and the cluster removing voltage is 100 eV; when the liquid chromatography-mass spectrometry is used for detecting the phosphatidylinositol ions in the solution to be detected, the collision voltage in the mass spectrometry condition is 50eV, and the declustering voltage is 70 eV.
Compared with the prior art, the invention has the following characteristics:
(1) according to the method, the non-targeted screening of the EPA and DHA type structure phospholipid in the krill oil is realized by a mode of combining precursor ion scanning and liquid chromatography-mass spectrometry, and compared with the existing liquid chromatography-mass spectrometry, a shotgun method and a rapid evaporation-ionization mass spectrometry, the detection molecular species of the EPA type structure phospholipid and the DHA type structure phospholipid can be effectively improved, so that 33 EPA and DHA type phospholipid molecular species can be simultaneously detected; meanwhile, the interference caused by matrix effect and ion inhibition in the detection process can be reduced, and the detection accuracy and integrity of the invention are improved;
(2) according to the invention, the secondary mass spectrum characteristics of EPA type structure phospholipid and DHA type structure phospholipid are researched, and are crushed by collision induced dissociation in a liquid chromatography-mass spectrometry, the ion strength of EPA and DHA is relatively outstanding when the mass-to-charge ratios of m/z301.2 and m/z327.2 are respectively measured, and on the basis, the separation effect of each phospholipid ion is further improved by optimizing the EPA type structure phospholipid and DHA type structure phospholipid mass spectrum methods, so that the detection accuracy and resolution are improved;
(3) the existing mass spectrometry detection method does not have specific detection process and parameters aiming at EPA-type and DHA-type structure phospholipids in food-borne materials, but the invention discovers that the energy degree deposited in ions before collision can be changed by voltage in the collision mass spectrometry, so that the strength of fragment ions is increased, the dissociation efficiency of EPA chains and DHA chains is improved, and the efficiency and the accuracy of the mass spectrometry in detection are improved; the change of the declustering voltage can accelerate ions, and naked analyte ions are generated through the collision of the ions and neutral molecules so as to eliminate additive solvent molecules and prevent the ions from aggregating together, thereby reducing the fluctuation of ion signals; on the basis, the invention respectively optimizes the collision voltage and the declustering voltage aiming at each phospholipid molecule, effectively relieves the influence on the signal response of ions caused by too low declustering voltage, and avoids the excessive fragmentation of the ions in the hole separator area in the source area caused by too high declustering voltage, thereby further improving the detection accuracy of the EPA and DHA type structure phospholipids;
(4) aiming at the phospholipid detection method combining precursor ion scanning and liquid chromatography-mass spectrometry, the invention also specifically optimizes the extraction method of the krill oil to-be-detected liquid, thereby greatly reducing the oxidation risk of lipid in the extraction process and further improving the accuracy and detection efficiency of the detection result;
therefore, the method can realize non-targeted screening of phospholipid in krill oil, and has the characteristics of high detection efficiency, high detection accuracy and high resolution.
Drawings
FIG. 1 is a spectrum of precursor ions of phosphatidylcholine ions in a phospholipid standard solution;
FIG. 2 is a precursor ion spectrum for phosphatidylethanolamine ion;
FIG. 3 is a precursor ion spectrum of phosphatidylinositol ion;
FIG. 4 is a graph of EPA chain dissociation efficiency for phosphatidylcholine ions;
FIG. 5 is a graph showing EPA chain dissociation efficiency for phosphatidylethanolamine ion;
FIG. 6 is a graph of EPA chain dissociation efficiency for phosphatidylinositol ions;
FIG. 7 is a graph of DHA chain dissociation efficiency for phosphatidylcholine ions;
FIG. 8 is a graph of DHA chain dissociation efficiency for phosphatidylethanolamine ion;
FIG. 9 is a graph of DHA chain dissociation efficiency for phosphatidylinositol ions;
FIG. 10 is a total ion flux chromatogram of EPA and DHA bound structural phospholipids in precursor ion scan mode;
FIG. 11 is a total ion flux chromatogram of an EPA-type structural phospholipid in precursor ion scan mode;
FIG. 12 is a total ion flow chromatogram of a DHA-type structural phospholipid in a precursor ion scan mode;
FIG. 13 is a mass spectrum of phosphatidylcholine ions in EPA-type structural phospholipids;
FIG. 14 is a mass spectrum of phosphatidylethanolamine ion in EPA-type structural phospholipids;
FIG. 15 is a mass spectrum of phosphatidylinositol ions in EPA-type structural phospholipids;
FIG. 16 is a mass spectrum of phosphatidylcholine ions in DHA-type structural phospholipids;
FIG. 17 is a mass spectrum of phosphatidylethanolamine ion in DHA-type structural phospholipids;
FIG. 18 is a mass spectrum of phosphatidylinositol ions in DHA-type structural phospholipids.
Detailed Description
The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.
Examples are given. An analytical detection method for effectively screening phospholipids in krill oil comprises the following steps:
taking 10g of krill meal, grinding the krill meal evenly, and adding the milled krill meal into 200mL of chloroform-methanol mixed solution, wherein the chloroform in the chloroform-methanol mixed solution is as follows: the volume ratio (v/v) of methanol is 2:1, and then the mixture is shaken for 2 minutes at room temperature to obtain an extracting solution A;
secondly, performing ultrasonic treatment on the A extracting solution to release krill oil from a solid mechanism to obtain a B1 mixture, centrifuging the B1 mixture, and collecting a lower organic phase and a centrifuged residue to obtain a B organic phase and a C residue;
thirdly, re-extracting the C residues twice by using 200mL of trichloromethane, and collecting a lower organic phase to obtain a D organic phase;
mixing the organic phase B and the organic phase D to obtain an E1 extract, then placing the E1 extract under a nitrogen blowing instrument for drying, then fixing the volume with acetonitrile, and then filtering through an organic filter membrane to obtain a solution to be detected E;
and fifthly, detecting the solution to be detected E by a hydrophilic interaction liquid chromatography-mass spectrometry combined method in a Precursor Ion Scanning (PIS) mode.
The concrete steps of the second step are as follows: ultrasonically extracting the A extractive solution at 20kHz and 130W for 15min to obtain B1 mixture, and centrifuging the B1 mixture at 4 deg.C at 8000g for 10min to obtain B organic phase and C residue.
The specific steps of the fourth step are as follows: and mixing the organic phase B and the organic phase D to obtain an E1 extract, blowing the E1 extract at the ambient temperature of 4 ℃ by using nitrogen flow, then fixing the volume by using 1mL of acetonitrile, and passing through a 0.22 mu m organic filter membrane to obtain a solution to be detected, wherein the solution to be detected is E.
The liquid chromatography conditions of the liquid chromatography-mass spectrometry in the fifth step are as follows: the liquid chromatographic column is a Cosmosil-HILIC chromatographic column (250X 4.6mm, particle size 3 μm); the sample introduction amount is 2 mu L, and the temperature of the chromatographic column is kept at 30 ℃; the mobile phase A is obtained by adding pure water into a mixed solution of 20mmol of ammonium formate and 20mmol of formic acid and fixing the volume to 1L; the mobile phase B is a formic acid acetonitrile solution with the concentration of 20mmol of the prepared formic acid.
The liquid chromatographic column elution method of the liquid chromatographic mass spectrometry in the fifth step specifically comprises the following steps: when the time is 0-10 min, the content of the mobile phase B is uniformly reduced from 95% to 70%, when the time is 10-15 min, the content of the mobile phase B is uniformly reduced from 70% to 50%, when the time is 15-20 min, the content of the mobile phase B is kept at 50% (the volume percent), and the balance is the mobile phase A in each time period in elution; after all lipids were eluted, the column was washed with 50% mobile phase B to adjust the HILIC conditions to the initial conditions of equilibrium, and the washed column was reusable.
The mass spectrum measuring mode of the liquid chromatography-mass spectrometry in the fifth step specifically comprises the following steps: injecting the E to-be-detected liquid as a sample into an electrospray ionization source by using the sample volume of 2 mu L, scanning precursor ions in a negative ion mode, and setting product ions of EPA-type structure phospholipid and DHA-type structure phospholipid as m/z301 and m/z327 respectively.
The mass spectrum conditions of the liquid chromatography-mass spectrometry in the fifth step are as follows: setting the ion spray voltage at 4500V in the negative ion mode, the ion source temperature was maintained at 500 ℃; the curtain gas, the drying gas (GS1), and the atomizer gas (GS2) in the ion source gas were set to 35psi, 50psi, and 60psi, respectively; the mass spectrum measurement range is m/z 450-950, and the scanning speed is 1s per spectrum; instrument control and data acquisition, processing and analysis were then performed using analyst1.6 software.
In the fifth step, when the liquid chromatography-mass spectrometry is used for detecting phosphatidylcholine ions (PC ions) in the solution to be detected, the collision voltage (CE) in the mass spectrometry condition is 50eV, and the declustering voltage (DP) is 100 eV; when the liquid chromatography-mass spectrometry is used for detecting phosphatidylethanolamine ions (PE ions) in a solution to be detected, the collision voltage in the mass spectrometry condition is 40eV, and the cluster removing voltage is 100 eV; in the liquid chromatography-mass spectrometry, when phosphatidylinositol ions (PI ions) in a solution to be detected are detected, the collision voltage in the mass spectrometry condition is 50eV, and the declustering voltage is 70 eV.
Experimental example 1: preparing phospholipid standard solution according to the method of the invention and detecting, wherein the phospholipid standard solution is detected after being crushed by using an induced collision dissociation (CID) method in a liquid chromatography-mass spectrometry method. As shown in the detection results of FIGS. 1-3, when the product ionic strength of the EPA-type structural phospholipid and the DHA-type structural phospholipid is m/z301.2 and m/z327.2, the EPA-type structural phospholipid and the DHA-type structural phospholipid can be stably screened in a non-targeted manner by a precursor ion scanning-liquid chromatography mass spectrometry method.
During detection, a collision voltage and a declustering voltage in a mass spectrum condition are respectively subjected to an orthogonal test, and then the dissociation efficiency of EPA chains and DHA chains in each phospholipid is detected. As shown in fig. 4 to 9, the dissociation efficiencies of EPA chain and DHA chain of PC, PE, PI ions were the best when the collision voltages were 50, 40, and 50eV, respectively; the EPA and DHA chain dissociation efficiencies of the ions of PC, PE and PI are best when the declustering voltages are 100, 100 and 70eV, respectively.
EPA and DHA conjugated structured Phospholipid (PL) in precursor ion scanning modeEPA/DHA) EPA type structural Phospholipids (PL)EPA) And DHA type structural Phospholipids (PL)DHA) The total ion current chromatograms (TIC) of (1) are shown in FIGS. 10-12, wherein PLEPA/DHAThe total ion flow chromatogram of (1) is shown in FIG. 10, PLEPA/DHARespectively has an elution order of PCE/D(11min)，PEE/D(12min)，PIE/DAnd PSE/D(16min), wherein PCE/DHas a peak strength significantly higher than that of PEE/DAnd PIE/D。
PLEPAThe total ion current chromatogram of (2) is shown in FIG. 11, and is related to PLEPA/DHASimilarly, it shows that PCE、PEEAnd PIEWidely available, but PCEThe signal intensity of (a) is only 2.8e6 cps.
PLDHAThe total ion current chromatogram is shown in FIG. 12, the chromatographic peak separation effect is good, and PC isDAnd PEDCompletely separated, but PCDHas a signal intensity (1.3e6cps) weaker than that of PCEAnd PI isDAlmost non-existent and not directly observable in the total ion flow pattern.
According to the method, the mode that precursor ion scanning and liquid chromatography-mass spectrometry are matched for detection is adopted, so that the phospholipid components in the krill oil can be effectively detected, and the method has good detection definition and accuracy.
PLEPA/DHAThe mass spectrum of each PL detected in krill oil is shown in FIGS. 13-18EPA/DHAThe chemical structure and content of the molecular species are shown in table 1. This application detects 8 PCs altogetherEThe molecular species, in which the ionic strength of m/z824.7 is greatest, was identified as PC 16: 0/20: 5, and in addition a phospholipid containing a double EPA chain, i.e.the ion m/z870.7(PC 20: 5/20: 5). At the same time, the plasmalogens with EPA structures are comprehensively detected, such as plasPC O-16: 0/20: 5(m/z810.7) and plasPC O-18: 0/20: 5(m/z 838.6). PE (polyethylene)EContains 5 molecular species, wherein the highest peak of m/z762.7 is identified as PE 18: 1/20: 5, while detecting plasPE O-16 of EPA type: 0/20: 5. the PEE contains 4 molecular species, of which m/z881.7(PI 18: 1/20: 5) and m/z855.7(PI 16: 0/20: 5) are the most abundant ions.
Eight PCs were detected in DHA-type structural phospholipidsDWherein the content of phospholipid molecules with double DHA chain structure m/z922.6(PC 22: 6/22: 6) is the highest, and the phospholipid molecules also contain plasmalogen with DHA structure, such as m/z836.5(PC O-16: 0/22: 6) and m/z862.6(PC O-18: 1/22: 6). PE (polyethylene)DThe molecular structure of the molecular structure has 6 molecular species, and m/z788.6(PE 18: 1/22: 6) ions are most remarkable. PI (proportional integral)DContains only 2 molecular species including m/z879.8(PI 16: 1/22: 6) and m/z895.8(PI O-18: 0/22: 6).
From the above, the combined method of precursor ion scanning and liquid chromatography-mass spectrometry used in the invention can effectively identify 33 PLsEPA/DHAAnd a large amount of plasmalogens with EPA/DHA structures are detected according to molecular species, so that the comprehensive detection range and good detection definition are realized.
TABLE 1 Krill oil Each PL detectedEPA/DHAOf molecular speciesChemical structure and content
Experimental example 2: in the experimental example, chromatographic profiles and retention times of an empty sample and a chemical standard solution are compared, a correction curve of each phospholipid under a series of concentration gradients is established, and a relation graph of a peak area and an analyte concentration is drawn by using a weighting factor of 1/x. As shown in Table 2, the calibration curve was linear over the concentration range of LOQ to 1000. mu.g/mL for each phospholipid standard, with an average correlation coefficient of 0.9978-0.9993, indicating that the peak area (y) and the phospholipid concentration (x,. mu.g/mL) are in good linear relationship. Determining the detection Limit (LOD) and quantification Limit (LOQ), PL of the measured object according to the signal-to-noise ratio (S/N) of 3 times and 10 times respectively by standard addition methodEPA/DHAThe LOD is less than or equal to 4.02 mu g/mL, and the LOQ is less than or equal to 13.4 mu g/mL, and the result shows that the detection method has higher sensitivity and can be used for PL in krill oilEPA/DHAScreening and quantification of (3).
Adding a phospholipid standard substance into a sample matrix to prepare a standard sample with the concentration of 50 mu g/mL, and then carrying out precision and recovery rate tests according to the detection method and detection conditions of the invention, wherein the results show that the relative standard deviation of the daily precision is less than or equal to 4.71 percent, and the relative standard deviation of the daily precision is less than or equal to 6.24 percent, which indicates that the detection method of the invention is used for detecting PLEPA/DHAThe result is accurate and the precision is high. Comparing the spiked sample with the standard prepared from the blank solution at 100% recovery shows that the method can recover PC, PE and PI with EPA/DHA structure well, with recovery ranging from 78.9% to 91.4% (RSD from 2.99% to 5.25%). No significant difference was observed between the phospholipids of EPA and DHA structure.
TABLE 2 verification of linearity, sensitivity, precision and recovery of the precursor ion scanning-liquid chromatography-mass spectrometry combination
Experimental example 3: in this experimental example, comparison tests were carried out by the precursor ion scanning-liquid chromatography mass spectrometry combination method of the present invention with conventional liquid chromatography mass spectrometry, a shotgun method and a rapid evaporation ionization mass spectrometry, respectively. The test results are shown in Table 3, and 33 kinds of PL can be detected by the precursor ion scanning-liquid chromatography-mass spectrometry combined methodEPA/DHAMolecular species, including 16 PCsE/D11 kinds of PEE/DAnd 6 kinds of PIE/D. Total detection of 11 PCs by normal phase liquid chromatography-mass spectrometryE/DHowever, since the system is in the positive ion mode, no phospholipid species of PE and PI were found. Rapid evaporation ionization mass spectrometry method for co-detection of 13 PLsEPA/DHAMolecule of which PCE/DOr PEE/DEach 6 kinds of, PI E/D6 kinds of, PAE/D1 kind of the Chinese medicinal composition. Shotgun method for detecting 15 kinds of PCE/DA molecule. As can be seen from the above, the combined scanning of precursor ions and liquid chromatography-mass spectrometry used in the present invention detects PL as compared with other methodsEPA/DHAThe molecular species are more, the plasmalogens with EPA and DHA structures can be comprehensively detected, and PL in a sample from a complex source can be screenedEPA/DHAHas higher superiority in the aspect.
Claims (8)
1. An analytical detection method for effectively screening phospholipids in krill oil is characterized by comprising the following steps:
taking 10g of krill powder, grinding uniformly, adding 100-300 mL of chloroform-methanol mixed solution, and oscillating for 1-3 minutes at room temperature to obtain an extracting solution A;
secondly, performing ultrasonic treatment on the A extracting solution to release krill oil from a solid mechanism to obtain a B1 mixture, centrifuging the B1 mixture, and collecting a lower organic phase and a centrifuged residue to obtain a B organic phase and a C residue;
thirdly, re-extracting the C residues for multiple times by using 100-300 mL of trichloromethane, and collecting a lower organic phase to obtain a D organic phase;
mixing the organic phase B and the organic phase D to obtain an E1 extract, blow-drying the E1 extract, fixing the volume with acetonitrile, and filtering with an organic filter membrane to obtain a solution to be detected E;
and fifthly, detecting the solution to be detected E by a hydrophilic interaction liquid chromatography-mass spectrometry combined method in a precursor ion scanning mode.
2. The method for analyzing and detecting phospholipids in krill oil effectively according to claim 1, wherein the step (II) comprises the following specific steps: and (3) performing ultrasonic-assisted extraction on the A extracting solution for 10-20 min under the conditions of 20kHz and 130W to obtain a B1 mixture, and then centrifuging the B1 mixture at 3-5 ℃ for 8-12 min by 8000g to obtain a B organic phase and a C residue.
3. The analytical detection method for efficiently screening phospholipids in krill oil according to claim 1, wherein the specific steps of the step (iv) are as follows: and mixing the organic phase B and the organic phase D to obtain an E1 extract, blowing the E1 extract at the ambient temperature of 4 ℃ by using nitrogen flow, then fixing the volume by using 1mL of acetonitrile, and passing through a 0.22 mu m organic filter membrane to obtain a solution to be detected, wherein the solution to be detected is E.
4. The analytical detection method for efficiently screening phospholipids in krill oil according to claim 1, wherein the liquid chromatography mass spectrometry in the fifth step is performed under the following liquid chromatography conditions: the liquid chromatographic column is a Cosmosil-HILIC chromatographic column; the sample introduction amount is 2 mu L, and the temperature of the chromatographic column is kept at 30 ℃; the mobile phase A is obtained by adding pure water into a mixed solution of 20mmol of ammonium formate and 20mmol of formic acid and fixing the volume to 1L; the mobile phase B is a formic acid acetonitrile solution with the concentration of 20mmol of the prepared formic acid.
5. The analytical detection method for effectively screening phospholipids in krill oil according to claim 1, wherein the liquid chromatography column elution method of the liquid chromatography-mass spectrometry in the fifth step specifically comprises: when the time is 0-10 min, the content of the mobile phase B is uniformly reduced from 95% to 70%, when the time is 10-15 min, the content of the mobile phase B is uniformly reduced from 70% to 50%, and when the time is 15-20 min, the content of the mobile phase B is kept at 50%.
6. The analytical detection method for effectively screening phospholipids in krill oil according to claim 1, wherein the mass spectrometry mode of the liquid chromatography-mass spectrometry method in the fifth step is specifically as follows: injecting the solution to be tested into an electrospray ionization source by the sample injection amount of 2 mu L, scanning precursor ions in a negative ion mode, and setting product ions of EPA-type structure phospholipid and DHA-type structure phospholipid as m/z301 and m/z327 respectively.
7. The analytical detection method for effectively screening phospholipids in krill oil according to claim 1, wherein the mass spectrometry conditions of the liquid chromatography-mass spectrometry in the fifth step are specifically as follows: setting the ion spray voltage at 4500V in the negative ion mode, the ion source temperature was maintained at 500 ℃; the gas curtain gas, the drying gas and the atomizer gas in the ion source gas are respectively set to be 35psi, 50psi and 60 psi; the mass spectrum measurement range is m/z 450-950, and the scanning speed is 1s per spectrum; data acquisition, processing and analysis are then completed.
8. The analytical detection method for effectively screening phospholipids in krill oil according to claim 1, wherein the liquid chromatography-mass spectrometry in the fifth step is characterized in that when phosphatidylcholine ions in the test solution are detected, the collision voltage is 50eV and the declustering voltage is 100eV under the mass spectrometry conditions; when the liquid chromatography-mass spectrometry is used for detecting phosphatidylethanolamine ions in a solution to be detected, the collision voltage in the mass spectrometry condition is 40eV, and the cluster removing voltage is 100 eV; when the liquid chromatography-mass spectrometry is used for detecting the phosphatidylinositol ions in the solution to be detected, the collision voltage in the mass spectrometry condition is 50eV, and the declustering voltage is 70 eV.
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