CN108828051B - Method for detecting lipid of antarctic krill oil in real time by rapid evaporation ionization mass spectrometry - Google Patents
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Abstract
The invention discloses a method for detecting the lipid of antarctic krill oil in real time by fast evaporation ionization mass spectrometry. The method comprises the following steps: A. extracting Antarctic krill oil with ethanol; B. after being ionized, the euphausia superba oil is introduced into a mass spectrometer for detection, and a mass spectrum profile graph is obtained; C. and (4) determining the lipidomics profile of the sample after MassLynx alignment of the mass spectrum profile map. The method does not need sample pretreatment, can realize real-time mass spectrometry detection, is suitable for lipidomics detection of food, and can promote the development of the shrimp sauce taking the Antarctic krill as the raw material.
Description
Technical Field
The invention relates to a method for detecting lipid of antarctic krill oil, in particular to a method for detecting lipid of antarctic krill oil in real time by fast evaporation ionization mass spectrometry.
Background
Lipidomics (Lipidomics) is a hot field which develops rapidly after genomics and proteomics, and research contents comprise structural identification and quantification of lipid molecular species in organisms and role analysis of the lipid molecular species in biological metabolism, diseases, immunity and the like. With the development of soft ionization technology and high-resolution mass spectrometry technology and the application thereof in lipid analysis, the rapid and high-precision analysis and detection of various trace lipids in a biological sample are realized, and the lipid analysis with scale and integrity is greatly promoted. In recent years, mass spectrometry has become more widely used, and researchers have made higher demands on high-throughput rapid mass spectrometry detection technology. However, biological tissue samples cannot be directly used for high-sensitivity mass spectrometry, and professionals are required to perform complex and tedious sample pretreatment steps such as tissue homogenization, compound extraction, separation and purification, and the like, so that the biological tissue samples can not be used for mass spectrometry, and therefore, the real-time mass spectrometry detection research of the tissue samples can not be realized, and the requirements of high-throughput omics technical research can not be met. The extraction methods adopted conventionally in lipidomic detection are a Folch method and a Bligh & Dyer method, however, the two methods need organic reagents with higher toxicity levels such as chloroform and the like, and are not suitable for lipidomic detection of food.
Rapid Evaporative Ionization Mass Spectrometry (REIMS) is a new technical means for in-situ dynamic real-time identification of clinical biological tissues, and is based on thermal ablation of biological tissue substances to induce ionization of biological molecules to form aerosol containing charged particles, which enters a mass spectrometer for analysis through an ion transmission pipeline. In 2009, Takatas et al developed REIMS based on a high-frequency electrotome device coupled high-resolution mass spectrometer on the basis of Desorption electrospray ionization (DESI), and provided a brand-new research method and thought for in-situ ionization mass spectrometry and mass spectrometry metabonomics.
Antarctic krill (Euphausia superba) is a class of crustacean zooplankton living in the south oceanic, has the characteristics of large biological storage amount, wide distribution and the like, and is an important strategic marine new resource. It is estimated that the storage capacity of the Antarctic krill is 6.5-10 million tons, and the maximum resistant fishing capacity is about 400-600 million tons. Under the background that world marine fishery resources are increasingly attenuated, research and development of antarctic krill are successively added in countries such as Japan and Norway, and related products such as krill oil are already industrialized. The antarctic krill is rich in phospholipid-bound n-3 fatty acid, is easier to absorb than triglyceride-bound n-3 fatty acid in fish oil, and is an effective source of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Experiments show that the euphausia superba oil has the physiological functions of reducing cholesterol, preventing senile dementia, preventing arteriosclerosis, resisting inflammation, protecting eyesight, improving brain learning function and the like. At present, research on Euphausia superba lipidomics is not reported, and development of the research on the Euphausia superba lipidomics can deepen the understanding of the nutrition of the Euphausia superba lipids and promote the development of the shrimp oil taking the Euphausia superba as the raw material.
Therefore, the existing lipidomics contour detection method has the problems of complicated sample pretreatment, incapability of realizing real-time mass spectrum detection and unsuitability for lipidomics detection of food; at present, lipidomics contour detection aiming at antarctic krill oil is not reported yet.
Disclosure of Invention
The invention aims to provide a method for detecting the lipid of antarctic krill oil in real time by using a rapid evaporation ionization mass spectrum. The invention can realize real-time mass spectrometric detection without sample pretreatment, and is suitable for lipidomics detection of food.
The technical scheme of the invention is as follows: the method for detecting the lipid of the antarctic krill oil by the rapid evaporation ionization mass spectrometry comprises the following steps:
A. extraction of Antarctic krill oil: homogenizing Antarctic krill, freeze-drying to obtain product A1, weighing product A1, placing into a centrifuge tube, adding ethanol into the centrifuge tube, shaking, mixing, leaching at normal temperature in a constant-temperature water bath kettle, centrifuging with a refrigerated centrifuge, transferring supernatant, adding ethanol into the lower layer precipitate, repeatedly extracting for two times, mixing supernatants, and evaporating ethanol to dryness at 65 ℃ to obtain oil concentrated solution, namely product A2;
B. ionizing product A2 by a heating probe to form aerosol, wherein the temperature of the heating probe is 500 ℃, the formed aerosol is driven by a Venturi pump through nitrogen at a pressure of 2bar, is introduced into a mass spectrometer through a PTFE tube in an orthogonal mode, and is injected into an injector introduction port through a needle pump by taking a propylene glycol/leucine enkephalin mixture as an auxiliary solvent; a Kanthal A1 filament of 1.1 omega, 3V and 500 ℃ is arranged in the mass spectrum interface and used as an auxiliary collision ionization, and ions enter a StepWave device of a mass spectrometer after collision; the scanning time of the mass spectrometer is 1 second, and the scanning range is m/z 50-1200; collecting data in a negative ionization mode to obtain a profile map of the antarctic krill oil mass spectrum;
C. and performing peak matching, peak alignment and noise filtering on the mass spectrum profile map by MassLynx, storing the result in a text format, obtaining the mass-to-charge ratio of a mass spectrum peak with the relative content of more than 5% and the fragment ion information of a main peak with the relative content of more than 10% according to mass spectrum data, and determining the lipidomics profile of the sample by combining with known data comparison.
In the method for detecting the lipid of the antarctic krill oil by the rapid evaporation ionization mass spectrometry, in the step a, the ethanol is 95% ethanol; adding 5ml of ethanol into each gram of sample into the centrifugal tube; centrifuging the centrifuge at 9000r/min for 10 min; the ethanol is added to the lower layer precipitation in an amount of 3ml of ethanol per gram of sample; the leaching time is 2 h.
In the method for detecting the lipid of the antarctic krill oil by the rapid evaporation ionization mass spectrometry, in the step a, the mass ratio of the propylene glycol to the leucine enkephalin is 98: 2.
In the method for detecting the lipid of the antarctic krill oil by the rapid evaporation ionization mass spectrometry, in the step B, the flow rate of the needle pump injected into the sample injector is 0.1 mL/min.
In the method for detecting the lipid of the antarctic krill oil by the rapid evaporation ionization mass spectrometry, in the step B, the product a2 is ionized by the heating probe, and the product a2 is diluted by 4 times with the pre-degummed soybean oil and then ionized by the heating probe.
Compared with the prior art, the method adopts REIMS high-flux real-time detection of lipidomics contour of the antarctic krill oil to realize the real-time detection of the lipid. The euphausia superba oil is ionized by an electrothermal probe or a single-stage electrode in a negative ionization mode to form aerosol, the formed aerosol is driven by a pump through nitrogen at a pressure of 2bar, the aerosol is introduced into a mass spectrometer through a PTFE tube in an orthogonal mode, parameters of the mass spectrometer are set, the mass spectrometer can obtain a mass spectrum contour map only by scanning for 1 second, and the lipidomics contour of a sample can be determined by software comparison. The method has the advantages that the lipidomics contour is detected quickly and efficiently in real time, the related data can provide a theoretical basis for mass spectrometry workers, and related basis and reference are provided for marine functional food enterprises and departments. The antarctic krill oil is detected by a mass analyzer to detect 20 fatty acids and 26 phospholipid molecules, wherein EPA (m/z 301, 21.59%) and DHA (m/z 327, 14.44%) are main ion peaks in a mass spectrum. The method does not need sample pretreatment, can realize real-time mass spectrometry detection, is suitable for lipidomics detection of food, and can promote the development of the shrimp sauce taking the Antarctic krill as the raw material.
Drawings
FIG. 1 is a detection device for detecting the lipidomics profile of antarctic krill in real time at a high throughput by REIMS;
FIG. 2 shows the effect of ethanol concentration on the extraction yield of Antarctic krill oil;
FIG. 3 is a REIMS mass spectrum profile of Antarctic krill oil;
FIG. 4 is a fatty acid ion peak in the region of m/z 200-500 of an Antarctic krill lipidomics profile;
FIG. 5 is a phospholipid ion peak in the range of m/z 400-900 of the Euphausia superba lipidomics profile.
The reference numbers in the figures are: 1-controller, 2-electrothermal probe, 3-antarctic krill oil concentrate, 4-aerosol, 5-injection pump (i.e. needle pump injection injector), 6-mass spectrometer.
Detailed Description
The present invention will be further described with reference to the accompanying drawings, which are not intended as a limitation on the present invention.
The following are experimental examples of the present invention.
Examples of the experiments.
1.1 Main instruments and devices
Xevo G2-XS quadrupole time-of-flight mass spectrometer: products of Waters corporation, usa, equipped with a fast evaporating ion source; pump11 elite needle Pump injection injector: product of Harvard corporation, USA; milliplus 2150 ultrapure water treatment system: millipore corporation, USA; masslynx 4.1 data acquisition software and LiveID statistical analysis software: products of W aters, USA; others are common laboratory instruments and equipment.
1.2 Main materials and reagents
Euphausia superba: a product of Euphausia superba from Liaoyu, science and technology development Co., Ltd; methanol, acetonitrile, isopropanol: all are chromatographically pure, a product of the company Meker, Germany; formic acid: chromatographic purity, product of Tedia corporation, USA; leucine enkephalin: Sigma-Aldrich, USA; other reagents commonly used in laboratories are analytical grade.
1.3 test conditions
1.3.1 Antarctic krill oil extraction conditions, namely homogenizing tissue of Antarctic krill, freeze-drying, accurately weighing 5.0g of sample in a 50mL centrifuge tube, adding 25mL of 95% ethanol, shaking, uniformly mixing, and leaching for 2 hours at normal temperature in a constant-temperature water bath kettle; centrifuging the mixture with a refrigerated centrifuge at 9000r/min for 10min, transferring the supernatant with a pipette, adding 15mL of 95% ethanol to the lower layer precipitate, repeatedly extracting twice, and mixing the supernatants; evaporating the organic solvent to dryness at 65 deg.C with a rotary evaporator to obtain Euphausia superba oil concentrate.
1.3.2 Mass Spectrometry conditions Euphausia superba oil samples were ionized by a heating probe (500 ℃), the formed aerosol was driven by Venturi pump nitrogen (2bar) and introduced into the mass spectrum via PTFE tube in an orthogonal manner, the main apparatus is as shown in FIG. 1; taking a propylene glycol/leucine enkephalin mixture (the mass ratio of the propylene glycol to the leucine enkephalin is 98:2) as an auxiliary solvent, introducing the mixture into a port through a needle pump injection sample injector at a flow rate of 0.1mL/min, and cleaning impurities, improving signal intensity and locking quality correction; kanthal A1 filament (1.1 omega, 3V, 500 ℃) is arranged in the mass spectrum interface and used as auxiliary collision ionization, and ions enter a mass spectrometer StepWave device after collision; scanning time of a mass spectrometer: 1 second; scanning range: m/z is 50-1200; all data were collected in negative ionization mode.
1.4 statistical analysis
And (3) carrying out peak matching, peak alignment, noise filtering and the like on the original mass spectrogram through MassLynx, and storing the result into a text format. And obtaining the mass-to-charge ratio of a mass spectrum peak with the relative content of more than 5 percent, the fragment ion information of a main peak with the relative content of more than 10 percent and the like according to mass spectrum data, and determining or inferring the structure information of the Lipid by combining the comparison of the Lipid MS Predictor and the Lipid map database search standard and literature reports.
2 results
2.1 Euphausia superba oil extraction optimization
The extraction methods conventionally used in lipidomic analysis are the Folch method and the Bligh & Dyer method, however, the two methods require organic reagents with higher toxicity levels, such as chloroform and the like. Considering the safety index of the euphausia superba oil as a health food, ethanol is used as an extraction medium in the experiment to extract the euphausia superba oil from euphausia superba dry powder. The influence of ethanol solutions with different concentrations on the extraction rate of the antarctic krill oil is optimized in comparison. As shown in FIG. 2, the yield of Antarctic krill oil increased with increasing ethanol content, with 95% ethanol extraction being the best and with a decrease in yield when pure ethanol was used.
2.2 REIMS Lipomics profiling
The REIMS is a real-time mass spectrum method without any sample pretreatment, a mass spectrum profile is obtained in real time by direct surface ionization of an original sample, and after the profile is identified by software such as LiveID and the like, dimension reduction and modeling are carried out on a characteristic ion peak, so that real-time identification and accuracy scoring of an unknown sample can be realized.
The ion source commonly used in REIMS is a unipolar electrode that rapidly vaporizes and ionizes small molecules on the surface by cutting tissue at a given current and enters the mass spectrometer port via an ion transport line. Because the Antarctic krill oil is a liquid sample, a monopolar electrode cutting mode cannot be used, the Antarctic krill oil is heated and quickly evaporated in an electric heating probe mode in the experiment, and lipid compounds are ionized and enter a mass spectrometer along with aerosol. The antarctic krill oil surface was tapped using an electrothermal probe with the mass spectrometry scan range set at m/z 0-1200 and mass spectrometry data were recorded by MassLynx. As shown in FIG. 3, the lipid molecular ion peak of Antarctic krill mainly appears in the region of m/z 200-900 and consists of two distinct peak clusters, namely the fatty acid ion peak cluster of m/z 200-500 and the phospholipid ion peak cluster of m/z 600-900. From the relative content, the signal response of the fatty acid ion peak is significantly stronger than the signal intensity of the phospholipid ion peak, and the peak area of the fatty acid ion peak is about 7.6 times that of the phospholipid ion peak. One part of the source of the fatty acid ions is generated by ionizing free fatty acids in the antarctic krill oil, and the other part of the source of the fatty acid ions is generated by high-energy cracking of lipids such as glyceride, phospholipid, cholesterol ester and the like by an electrothermal probe, and the fragmented and ionized fatty acid chains are reflected in a mass spectrogram.
FIG. 4 is a fatty acid ion peak in a region of an antarctic krill lipidomics contour map m/z 200-500, wherein the separation degree of each peak is good, and a matrix peak is hardly seen due to high signal-to-noise ratio; the structures and relative contents obtained by identifying and integrating the compounds are shown in Table 1. The signal response intensity is the largest m/z 301, the relative content reaches 21.59 percent, and the structure is identified as FA 20:5, which is known as EPA. Second, m/z 327(FA 22:6/DHA, 14.44%), m/z 281(FA 18:1, 12.62%), and m/z 255(FA 16:0, 18.74%), respectively. The fatty acid ion peak m/z 275 (3.78%) of suspected FA 18:4 was detected in Antarctic krill oil, a component that is commonly detected in vegetable oil crops. Meanwhile, m/z 391, m/z 417 and the like are suspected to be ultra-long chain fatty acids FA 26:2 (6.17%) and FA 28:3 (2.10%), and the existence of the ultra-long chain fatty acids is possibly related to that antarctic krill is stressed by an extremely cold environment to promote the metabolism of the antarctic krill to generate long chain fatty acids so as to improve the fluidity of body fluid; the related hypothesis requires further biological validation.
TABLE 1 analysis of relative fatty acid content and structure in Antarctic krill oil
Note: 1) the fatty acid structure is the result analyzed according to experience and software, and the structure verification is not carried out; the structure is represented by FA c: d, wherein c is the number of carbon atoms of the fatty acid chain and d is the number of double bonds.
FIG. 5 shows the phospholipid ion peak in the range of m/z 400-900, since the signal response of phospholipid is significantly lower than that of fatty acid, the matrix effect is relatively strong, and 26 phospholipid molecules are identified after noise reduction and peak extraction. Wherein the ion peak with the strongest signal is m/z 763 (11.67%), and the structure is suspected to be [ PG 36:7-H ]]－(ii) a Followed by m/z 737 (11.46%), structure [ PC o-38:3-choline ]]－/[PE o-38:3-NH3]－. The molecular structure of PC with missing choline fragments is the same as the deamination structure of PE at the same c: d ratio, hence in REIMSNo distinction in lipidomics profile spectra was possible. Taking mass spectrum peak m/z 695.4 as an example, the total carbon atoms and double chain number of sn1/sn2 fatty acid chain is identified to be 34:3, and the molecular structure composition of the fatty acid chain may be [ PC-choleline ]]–Or [ PE-NH3]–Or a superposition of both. The Antarctic krill oil has fewer PI molecules and only m/z 835([ PI 34: 1-H)]－1.79%) and m/z 843([ PI o-36: 4-H)]－1.47%) was detected.
TABLE 2 analysis of relative phospholipid content and structure in Antarctic krill oil
2.3 methodological validation
The organic solvent has a low ignition point, and is easy to generate open fire through ionization of an electric heating probe, and a standard quantitative solvent system is not constructed at present. Therefore, the sensitivity test in the methodological validation adopts a multiple dilution method, and REIMS detection is carried out after dilution by adding pre-degummed soybean oil into the euphausia superba oil. The result shows that 26 phospholipid molecules can still be rapidly detected after the antarctic krill oil is diluted by 4 times, the spectrogram is similar to that in the figure 5 after noise reduction, and lipidomics contour characteristics are similar except that the signal intensity is reduced, and no significant difference exists; after the antarctic krill oil is diluted by 10 times, the number of detected phospholipid molecules is reduced to 14, the signal intensity of each phospholipid ion peak is reduced in an equal proportion, and partial contour features are lost. The precision test adopts the daily precision and the daytime precision evaluation,
fatty acid and phospholipid are respectively selected from two ions of m/z 301, m/z 327, m/z 763 and m/z 737, and are continuously measured for 7 times and 5 days in the same day respectively, and the daily precision RSD is calculated to be less than or equal to 6.24 percent and the daily precision RSD is calculated to be less than or equal to 8.57 percent. The methodological verification result shows that the method can meet the requirement of lipidomics contour analysis and test of fatty acid and phospholipid in the Antarctic krill oil.
3 conclusion
And (3) directly ionizing the lipid in the antarctic krill oil by adopting a REIMS technology and obtaining a real-time lipidomics profile. In the negative ion mode, the antarctic krill oil is excited into an ionic state by an electrothermal probe, is introduced into a mass spectrum port along with aerosol, and is detected by a mass analyzer for 20 fatty acids and 26 phospholipid molecules, wherein EPA (m/z 301, 21.59%) and DHA (m/z 327, 14.44%) are main ion peaks in a mass spectrum. The method is rapid and efficient in real-time detection of the Euphausia superba oil lipidomics contour, and relevant data can provide a theoretical basis for mass spectrometry workers and relevant basis and reference for marine functional food enterprises and departments.
The following are examples of the present invention.
Examples
The method for detecting the lipid of the antarctic krill oil by the rapid evaporation ionization mass spectrometry comprises the following steps:
A. extraction of Antarctic krill oil: homogenizing Antarctic krill, freeze-drying to obtain product A1, weighing product A1, placing into a centrifuge tube, adding ethanol into the centrifuge tube, shaking, mixing, leaching at normal temperature in a constant-temperature water bath kettle, centrifuging with a refrigerated centrifuge, transferring supernatant, adding ethanol into the lower layer precipitate, repeatedly extracting for two times, mixing supernatants, and evaporating ethanol to dryness at 65 ℃ to obtain oil concentrated solution, namely product A2;
B. ionizing product A2 by a heating probe to form aerosol, wherein the temperature of the heating probe is 500 ℃, the formed aerosol is driven by a Venturi pump through nitrogen at a pressure of 2bar, is introduced into a mass spectrometer through a PTFE tube in an orthogonal mode, and is injected into an injector introduction port through a needle pump by taking a propylene glycol/leucine enkephalin mixture as an auxiliary solvent; a Kanthal A1 filament of 1.1 omega, 3V and 500 ℃ is arranged in the mass spectrum interface and used as an auxiliary collision ionization, and ions enter a StepWave device of a mass spectrometer after collision; the scanning time of the mass spectrometer is 1 second, and the scanning range is m/z 50-1200; collecting data in a negative ionization mode to obtain a profile map of the antarctic krill oil mass spectrum;
C. and performing peak matching, peak alignment and noise filtering on the mass spectrum profile map by MassLynx, storing the result in a text format, obtaining the mass-to-charge ratio of a mass spectrum peak with the relative content of more than 5% and the fragment ion information of a main peak with the relative content of more than 10% according to mass spectrum data, and determining the lipidomics profile of the sample by combining with known data comparison.
In the step A, the ethanol is 95 percent ethanol; adding 5ml of ethanol into each gram of sample into the centrifugal tube; centrifuging the centrifuge at 9000r/min for 10 min; the ethanol is added to the lower layer precipitation in an amount of 3ml of ethanol per gram of sample; the leaching time is 2 h.
In the step A, the mass ratio of the propylene glycol to the leucine enkephalin is 98: 2.
In the step B, the flow rate of the needle pump injected into the injector is 0.1 mL/min.
In the step B, the A2 product is ionized by a heating probe after the A2 product is diluted by 4 times with the pre-degummed soybean oil.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it should be understood that various changes and modifications can be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (4)
1. The method for detecting the lipid of the antarctic krill oil by the rapid evaporation ionization mass spectrometry is characterized by comprising the following steps of:
A. extraction of Antarctic krill oil: homogenizing Antarctic krill, freeze-drying to obtain product A1, weighing product A1, placing into a centrifuge tube, adding ethanol into the centrifuge tube, shaking, mixing, leaching at normal temperature in a constant-temperature water bath kettle, centrifuging with a refrigerated centrifuge, transferring supernatant, adding ethanol into the lower layer precipitate, repeatedly extracting for two times, mixing supernatants, and evaporating ethanol to dryness at 65 ℃ to obtain oil concentrated solution, namely product A2;
B. ionizing product A2 by a heating probe to form aerosol, wherein the temperature of the heating probe is 500 ℃, the formed aerosol is driven by a Venturi pump through nitrogen at a pressure of 2bar, is introduced into a mass spectrometer through a PTFE tube in an orthogonal mode, and is injected into an injector introduction port through a needle pump by taking a propylene glycol/leucine enkephalin mixture as an auxiliary solvent; a Kanthal A1 filament of 1.1 omega, 3V and 500 ℃ is arranged in the mass spectrum interface and used as an auxiliary collision ionization, and ions enter a StepWave device of a mass spectrometer after collision; the scanning time of the mass spectrometer is 1 second, and the scanning range is m/z 50-1200; collecting data in a negative ionization mode to obtain a profile map of the antarctic krill oil mass spectrum;
C. performing peak matching, peak alignment and noise filtering on the mass spectrum profile map by MassLynx, storing the result in a text format, obtaining the mass-to-charge ratio of a mass spectrum peak with the relative content of more than 5% and the fragment ion information of a main peak with the relative content of more than 10% according to mass spectrum data, and determining the lipidomics profile of the sample by combining with known data comparison;
the A2 product is ionized by heating probe after diluting A2 product with pre-degummed soybean oil by 4 times.
2. The method for detecting the lipid of the antarctic krill oil by the fast evaporation ionization mass spectrometry according to claim 1, wherein the method comprises the following steps: in the step A, the ethanol is 95 percent ethanol; adding 5ml of ethanol into each gram of sample into the centrifugal tube; centrifuging the centrifuge at 9000r/min for 10 min; the ethanol is added to the lower layer precipitation in an amount of 3ml of ethanol per gram of sample; the leaching time is 2 h.
3. The method for detecting the lipid of the antarctic krill oil by the fast evaporation ionization mass spectrometry according to claim 1, wherein the method comprises the following steps: in the step A, the mass ratio of the propylene glycol to the leucine enkephalin is 98: 2.
4. The method for detecting the lipid of the antarctic krill oil by the fast evaporation ionization mass spectrometry according to claim 1, wherein the method comprises the following steps: in the step B, the flow rate of the needle pump injected into the injector is 0.1 mL/min.
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