CN114019039A - Method for selectively extracting phospholipid from plasma sample and phospholipid detection method - Google Patents
Method for selectively extracting phospholipid from plasma sample and phospholipid detection method Download PDFInfo
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- CN114019039A CN114019039A CN202111190612.8A CN202111190612A CN114019039A CN 114019039 A CN114019039 A CN 114019039A CN 202111190612 A CN202111190612 A CN 202111190612A CN 114019039 A CN114019039 A CN 114019039A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention discloses a method for selectively extracting phospholipid from a plasma sample and a phospholipid detection method, wherein the extraction method comprises the following steps: a. extracting the total lipid residue from the plasma; b. 10% by weight of TiO was used2the/KCC-1 core-shell microspheres are used as an adsorbent, and phospholipid in the total lipid is adsorbed by using a solid phase extraction method; c. the phospholipids are eluted from the adsorbent and dried under vacuum to give purified phospholipids. The method of the invention can be used for more rapidly refiningThe phospholipids are quasi-purified from plasma and further characterized and quantified.
Description
Technical Field
The invention relates to a method for selectively extracting phospholipid from a plasma sample and a phospholipid detection method, belonging to the technical field of biomedicine.
Background
Phospholipids are a class of amphipathic lipids whose molecules have a polar "head" comprising a phosphate group and two apolar fatty acid tails, linked by a glycerol molecule. In addition to being a major component of the cell membrane, phospholipids play an important role in many cellular processes. Serum phospholipids have been continuously shown to be involved in the etiology of several complex diseases including cardiovascular disease and cancer. Thus, as an important resource for polyunsaturated fatty acids, the availability of serum phospholipids derived from enzymes that ingest diet and control endogenous metabolism is of great significance to health. Recent developments in metabolomics technology provide new insights into the identification of plasma biomarkers for different diseases. There is increasing evidence that plasma phospholipid substances, including Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI), Phosphatidylserine (PS) and Sphingomyelin (SM), play an important role in different aspects of ischemic stroke. However, since lipid substances in plasma are complex and various and a high-precision and effective detection method is not available, direct association of phospholipids with health cannot be clarified in the current research.
In order to further study the potential role of phospholipids in specific diseases of human bodies, a more convenient and accurate phospholipid extraction method and a detection method are particularly important.
Disclosure of Invention
The invention aims to provide a method for selectively extracting phospholipid from a plasma sample and a phospholipid detection method. The method can purify the phospholipid from the plasma more rapidly and accurately, and further can perform characterization and quantitative detection on the phospholipid.
The technical scheme of the invention is as follows: a method for selectively extracting phospholipids from a plasma sample, comprising the steps of:
a. extracting the total lipid residue from the plasma;
b. 10% by weight of TiO was used2the/KCC-1 core-shell microspheres are used as an adsorbent, and phospholipid in the total lipid is adsorbed by using a solid phase extraction method;
c. the phospholipids are eluted from the adsorbent and dried under vacuum to give purified phospholipids.
In the above method for selectively extracting phospholipids from a plasma sample, the specific method of step a is:
mixing 1.5mL of chloroform/methanol solution at a volume ratio of 2:1 per 100. mu.L of plasma sample to obtain a mixture, subjecting the mixture to vortex oscillation for 2 minutes followed by sonication for 15 minutes, then centrifuging at 12000 g at 8 ℃ for 5 minutes to remove insoluble fractions from the mixture, repeating one vortex oscillation, sonication and centrifugation as before, adding 0.2mL of water to the mixture to induce phase separation, and separating to obtain a lower organic phase, and vacuum evaporating the lower organic phase for freeze-drying to obtain a total lipid residue, which is stored at-80 ℃ before further preparation.
In the aforementioned method for selectively extracting phospholipids from a plasma sample, the specific steps of step b are:
the SPE column is filled with 10 weight percent of TiO2the/KCC-1 core-shell microspheres were activated sequentially with methanol and 0.1% FA, the total lipid residue was passed through an SPE column with 100. mu.L of Folch solution (i.e., chloroform/methanol solution at 2:1 by volume), and after loading, the SPE column was washed with 2:1 by volume chloroform/2-propanol and 10% by mass aqueous methanol to remove fatty acids and all other non-phosphate group containing lipids.
In the aforementioned method for selectively extracting phospholipid from a plasma sample, the preparation method of the 10 wt% TiO2/KCC-1 core-shell microsphere comprises:
adding 1.10g of cetylpyridinium chloride and 0.60g per 30mL of water, adding 0.94mL of isopropanol and 30mL of cyclohexane with stirring, then, dropwise adding 3mL of ethyl n-silicate to the mixture using a syringe, vigorously stirring the homogeneous mixture at room temperature for 30 minutes, heating to 70 ℃, and holding for 20 hours, after the reaction, centrifuging the solution at 8000g for 10 minutes, washing the residue three times with acetone, deionized water, and ethanol in this order, then vacuum-drying for 12 hours, and finally, obtaining KCC-1 powder by removing the surfactant from the silica nanoparticles at 500 ℃ in a muffle furnace for 4 hours;
dispersing 2.20g of tetrabutyl titanate in 8.80mL of ethanol, sealing the solution and shaking for 5 minutes under vortex, then, adding the mixture to KCC-1 powder under stirring until dry, drying the solidThe bulk material was heated to 100 ℃ for 6 hours, then 1g of the heated solid material (silica sample carrying the titanium dioxide precursor) was placed in an autoclave containing 20mL of water and kept at 60 ℃ for 5 hours, and finally the dried sample was heat-treated in a muffle furnace at 600 ℃ for 5 hours to obtain 10% by weight of TiO2KCC-1 core-shell microspheres.
In the aforementioned method for selectively extracting phospholipids from a plasma sample, the specific steps of step c are:
the target phospholipid was eluted from the SPE cartridge using chloroform/methanol at a volume ratio of 1:2 and dried under vacuum, and the resulting phospholipid residue was redissolved in 500. mu.L of Folch solution and filtered through a 0.22. mu.m PTFE (Polytetrafluoroethylene) membrane before performing the H phospholipid C-MS analysis.
In the method for selectively extracting phospholipid from a plasma sample, the SPE column used in the solid phase extraction method in the step b is a monomer booster-type SPE column, and the structure of the SPE column comprises a column body, wherein one end of the column body is provided with a detachably connected pressure plug; the pressure plug comprises a cover body which is hermetically connected with the pipe orifice of the column body, the upper end of the cover body is provided with a hollow structure, a telescopic air chamber is arranged in the hollow structure, and the lower end of the telescopic air chamber is communicated with the column body through an air hole in the cover body; the upper end of the telescopic air chamber is provided with a pulling plate, and the telescopic air chamber is provided with a tension spring of which two ends are respectively connected with the pulling plate and the cover body; the two sides of the hollow structure are provided with sliding grooves, and the pull plate is provided with a handle which penetrates through the sliding grooves and protrudes out of the hollow structure.
In the aforementioned method for selectively extracting phospholipid from a plasma sample, the top of the hollow structure is provided with a rotating baffle plate; the upper end of the rotary baffle is provided with a knob, and the two ends of the rotary baffle are provided with lugs; the hollow structure is provided with a transverse limiting groove corresponding to the lug, so that the lug can rotate within an angle limited by the transverse limiting groove; the upper end of the handle is provided with a hook corresponding to the lug.
In the aforementioned method for selectively extracting phospholipid from a plasma sample, a connection lug is disposed at the tube opening of the cylinder, a sealing chute is disposed at the lower end of the cover body, a notch corresponding to the connection lug is disposed on the sealing chute, and a sealing ring is disposed in the sealing chute.
The phospholipid detection method based on the extraction method comprises the following steps: the selective extraction of phospholipids from plasma samples using the methods described previously resulted in purified phospholipids which were then characterized and quantitatively detected using hydrophilic interaction chromatography-mass spectrometry.
In the phospholipid detection method, the specific method for performing characterization and quantitative detection by the hydrophilic interaction chromatography-mass spectrometry method comprises the following steps:
performing chromatographic separation by using a Cosmosil HILIC chromatographic column at 30 ℃ by using a high performance liquid system, wherein ultrapure water containing 20mM ammonium formate and 0.1% formic acid is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the gradient elution flow rate was 0.6 mL. min-1(ii) a The final optimization conditions are as follows: 0.0 minute, 5% A-95% B; 3.0 min, 5% a-95% B; 4.0 min 12% A-88% B; 12% A-88% B at 10.0 min; 50% A-50% B at 12.0 min; 17.0 min 50% A-50% B; then placing the sample bottle in an autosampler at 8 ℃, injecting 1 mu L of purified phospholipid sample solution into an HPLC system for analysis, and carrying out mass spectrometry by using a triple quadrupole mass spectrometer and the HPLC system; the mass spectrometry apparatus was operated in negative ion mode using electrospray ionization method; the MS full scanning function is used for formal sample analysis, the scanning time is 1s, and the scanning mass range is m/z 600-1000; the sub-ion scanning function is used for tandem mass spectrometry, the optimized collision energy is 22-40V, and the scanning time is set to be 0.1 s; argon was used as the collision gas for MS/MS analysis; other general mass spectral parameters were as follows: capillary voltage, 4.0 KV; cone voltage, 30V; desolventizing temperature is 500 ℃; desolventizing air flow rate, 1000 L.h-1(ii) a Cone gas flow rate, 30 L.h-1。
Compared with the prior art, the invention synthesizes more effective and efficient TiO by extracting total lipid from plasma by a modified Folch method and then utilizing the principle of chelating bidentate bonds between phosphate groups and metal oxides2the/KCC-1 core-shell microsphere is used as a solid phase extraction adsorbent to adsorb phospholipid in total lipid, namely the TiO of the invention2the/KCC-1 core-shell microsphere is a microcosmic core-shell structure consisting of fiber nano titanium dioxide and nano silicon dioxide microspheres with extremely high porosity, and KCC-1 nano particles with mesoporesHas a corrugated structure, remarkably increases the surface area, and is coated with TiO2The extraction efficiency of phospholipid can be improved after the nano-fibers are adopted, so that the phospholipid can be selectively adsorbed from the total lipid more efficiently, the extraction method is quicker and more effective, and the purity of the extracted phospholipid is up to more than 98.5%. In addition, in order to match with the TiO2/KCC-1 core-shell microsphere adsorbent, the invention also uses an SPE column with a special structure, the SPE column is provided with a detachable pressure plug, and the pressure plug can provide stable forward pressure for the SPE column by utilizing the elasticity of a tension spring without using vacuum suction equipment. The pressure plug is simple and small in structure, convenient to use, reusable and free of occupying laboratory space.
Furthermore, the hydrophilic interaction chromatography-mass spectrometry method of the present invention, which is a normal phase chromatography method in which the phospholipids of interest are separated according to the polarity of their phosphate polar head, means that the phospholipids will elute from the HILIC column according to their class, and the more polar the phospholipids have a longer retention time, once the class is determined, different phospholipid molecules in the same class can be easily identified according to the length of the fatty acid chain and the degree of unsaturation.
Drawings
FIG. 1 is a representative mass spectrometric profile of the present invention for mass spectrometric detection of a plasma sample;
FIG. 2 is a schematic diagram of the single pressurized SPE column structure of the present invention;
FIG. 3 is a side view of FIG. 2;
FIG. 4 is a top view of FIG. 2;
fig. 5 is a bottom view of fig. 2.
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.
1. And (4) collecting a plasma sample.
Ethical approval was obtained from the research ethical committee of the first subsidiary hospital of the university of medical science of wenzhou (reference number: 2018-. Informed consent for the use of blood samples for scientific research purposes was obtained from recruited volunteers (>18 years).
2. And (4) preparing a sample.
Total lipids were extracted from human plasma using a modified Folch method. A specific method included extracting a portion of 100. mu.L of plasma with 1.5mL of Folch's solution in chloroform/methanol (2:1, v/v). The mixture was vortexed for 2 min, sonicated for 15 min, and then centrifuged at 12000 g for 5 min at 8 ℃ (Eppendorf 5424R, germany). When the insoluble fraction was removed, after another vortex mixing and centrifugation cycle, 0.2mL of water was added to the mixture to induce phase separation. The lower organic phase was carefully separated and lyophilized by evaporation in vacuo to give a total lipid residue which was stored at-80 ℃ prior to further preparation.
Laboratory synthetic filled TiO with Total lipid residue2A/KCC-1 SPE cartridge (10mg/1ml cartridge) was treated to selectively extract phospholipids with reference to the SPE method previously reported (Zhang et al, 2021). The SPE cartridge was activated initially with methanol and 0.1% FA. The total lipid residue was passed through the SPE column with the necessary amount of Folch solution. After loading, the SPE cartridge was washed with chloroform/2-propanol (2:1, v/v) and 10% aqueous methanol to remove fatty acids and all other phosphate group-free lipids. From TiO using chloroform/methanol (1:2, v/v)2The target phospholipid fraction was eluted on a/KCC-1 SPE cartridge and dried under vacuum. The phospholipid residue obtained was redissolved in 500. mu.L of Folch's solution and filtered through a 0.22 μm PTFE (Polytetrafluoroethylene) membrane before performing H phospholipid C-MS analysis.
3. And (3) performing high performance liquid chromatography-mass spectrometry.
Chromatographic separation was carried out using a High Performance Liquid Chromatography (HPLC) system (Waters, ACQUITY H-Class, Milford, MA, USA) at 30 ℃ using a Cosmosil HILIC column (5 μm, 150 mm. times.4.6 mm, Nacalai Tesque, Japan). The mobile phase A was ultrapure water containing 20mM ammonium formate and 0.1% formic acid, and the mobile phase B was acetonitrile. The gradient elution flow rate was 0.6mL min-1, and the final optimization conditions were as follows: 0.0 minute, 5% A-95% B; 3.0 min, 5% a-95% B; 4.0 min 12% A-88% B; 12% A-88% B at 10.0 min; 50% A-50% B at 12.0 min; 17.0 min 50% A-50% B. The sample vial was placed in an autosampler at 8 ℃ and 1 μ L of the sample solution was injected into the HPLC system for analysis.
Triple quadrupole mass spectrometers (Waters, Xevo TQD, Manchester, UK) were used in conjunction with HPLC systems for mass spectrometry. The mass spectrometry apparatus was operated in negative ion mode using electrospray ionization (ESI) method. The MS full scanning function is used for formal sample analysis, the scanning time is 1s, and the scanning mass range is m/z 600-1000. The sub-ion scanning function is used for tandem mass spectrometry (MS/MS), the optimized collision energy is 22-40V, and the scanning time is set to be 0.1 s. Argon was used as the collision gas for MS/MS analysis. Other general mass spectral parameters were as follows: capillary voltage, 4.0 KV; cone voltage, 30V; desolventizing temperature is 500 ℃; desolventizing air flow rate, 1000L h-1(ii) a Conical gas flow rate, 30L h-1。
4. And (4) data acquisition and statistical analysis.
All HPLC-MS data were collected using MassLynx V4.1 software (Waters, UK). Statistical analysis was performed using Microsoft Office Excel (2007, usa), SIMCA (version 14.1, MKS unmetrics, sweden), and SPSS Statistics (version 19, IBM corporation, usa).
To analyze the differences between the three groups, one-way analysis of variance (ANOVA) was performed with a significance level of 0.05. The Levene test is used to evaluate the equality of the variances. If the variances between the three groups are equal (homogeneity of variance), the Least Significant Difference (LSD) is calculated to check for statistical significance. If the variances between the three groups are not equal, a Tamhane's T2 test was performed to check for statistical significance. A P value of 0.05 or less indicates that the difference is statistically significant, while a P value >0.05 indicates that the difference is not statistically significant.
5. Identification and quantification of phospholipids.
In order to selectively perform phospholipid analysis using LC-MS, a collected plasma sample should be extracted using a special protocol. The traditional lipid extraction method is Folch, Bligh&Dyer and methyl tert-butyl ether (MTBE) (Ranjith Kumar et al, 2015). According to some previous studies, the Folch method was considered most suitable for extracting various lipid classes from biological fluid samples (Ranjith Kumar et al, 2015; Ulmer et al, 2018).Thus, the Folch method was initially selected and optimized to extract total lipids from human plasma. Unlike other neutral lipids such as cholesterol esters and triglycerides, all phospholipid molecules have a unique phosphate group that can react with TiO2And (4) combining. As the mesoporous KCC-1 nano-particles have a wrinkle structure, the surface area is obviously increased, and the surface is coated with TiO2To improve the efficiency of phospholipid extraction. TiO for efficient phospholipid extraction2the/KCC-1 material was synthesized using the method we previously reported (Zhang et al, 2021)). For this purpose, the chromatographic material TiO synthesized using the aforementioned method2the/KCC-1 core shell microspheres were further treated with the total lipid residue obtained by SPE method to separate plasma phospholipids from the total lipid.
To facilitate the analysis of phospholipids in human plasma, we used a dedicated triazole-HILIC column for chromatographic separation. As a normal phase chromatography method, the phospholipids of interest are separated according to the polarity of their phosphate polar head, which means that the phospholipids will elute from the HILIC column according to their class, and the more polar the phospholipids have longer retention times. Once the class is determined, different phospholipid molecules in the same class can be easily identified based on the length and unsaturation of the fatty acid chains. Human plasma phospholipids PC, PE, Sphingomyelin (SM) and PI elute from the used HILIC column according to the polarity in the Total Ion Chromatogram (TIC) with reference to the retention time of the corresponding phospholipid standards. SM, also known as sphingomyelin, as the major component of the cytoplasmic membrane is a unique member of the phospholipid family, in which glycerol is replaced by sphingosine, the C-1 alcohol group of which is esterified to phosphorylcholine. The corresponding mass spectra obtained for each phospholipid class in negative ionization mode with MS are shown in figure 1. Individuals in each phospholipid class were identified by their fragment ions of the fatty acid chains in the tandem mass spectrometry (MS/MS) spectrum. For example, the PC peak at 8.79 minutes the dominant ion m/z 802.7 produced two fatty acid chain fragment ions at m/z 255.2 (palmitic acid, FA16:0) and m/z 279.7 (linoleic acid, FA 18). Thus M/z 802.7 was determined to be the formate adduct [ M + COOH ] of PC (16:0/18:2)]-. The corresponding ions at M/z 742.8 and M/z 792.6 were identified as their demethylated ions [ M-CH ] according to their MS/MS spectra and M/z value differences, respectively3]-And chloride ion adduct [ M + Cl ]]-。
In addition to normal PE, four Plasmalogens (PE) (p-14:0/20:4), PE (p-16:1/20:4), PE (p-16:0/20:4) and PE (p-16:0/22:6) were also detected at 9.23 minutes. Plasmalogens are a key phospholipid that play a role in human health by participating in neuronal development and immune responses and acting as an endogenous antioxidant. Plasmalogens generally consist of a fatty alcohol having a vinyl ether bond at the sn-1 position and a polyunsaturated fatty acid at the sn-2 position of the glycerol backbone. Plasmalogens are mainly classified into PC plasmalogens and PE plasmalogens in terms of their head group. The retention time of the same phospholipid molecule is mainly determined by the length of the fatty acid chain, and the retention time is shortened along with the increase of the length of the phospholipid chain. Thus, two similar peaks at 9.76 and 10.18 minutes in TIC can be attributed to SM class. In the MS spectra, SM (d18:1/22:1), SM (d18:1/22:0), SM (d18:1/24:2), SM and (d18:1/24:1) have longer fatty acid chains that elute from the column at 9.76 minutes, while SM (d18:1/16:1), SM (d18:1/16:0), SM (d18:1/18:1) and SM (d18:1/18:0) shorter fatty acid chains elute at 10.18 minutes. These results are consistent with previously reported conclusions. At the end of the HILIC separation, PI was detected at 14.52 min. In the MS spectrum, the main peak at M/z 885.6 was identified as the deprotonated ion [ M-H ] of PI (18:0/20:4)]-. In summary, the detection method extracts and identifies 31 phospholipids from human plasma, including 7 PCs, 10 PEs, 8 SMs and 6 PIs.
Ionization efficiency of different covalently linked compounds in electrospray ionization mass spectrometry (ESI-MS) depends on the dipole potential present in the zwitterionic polar head of phospholipid molecules (Han and Gross, 2005). Since the polar head portion of the phospholipid molecule constitutes the dominant dipole of the whole molecule, the ionization efficiency of different phospholipid molecules of the same class is the same under the same experimental conditions. For example, all PCs have the same ionization efficiency as the commercially available PC standard. Thus, a weighted linear regression model can be used to construct a PC quantified calibration curve using external PC standards. Calibration curves for four standards, PC (18:0/20:4), PE (18:0/18:0), SM (d18:1/24:0), and PI (18:0/20:4) Using an extracted ion chromatogram (X)IC) the peak area of the quantified ions versus analyte concentration. LLOQ (lower limit of quantitation) is based on signal-to-noise ratio (S/N)>10) And (4) defining. Quantitative ion, calibration curve, linear correlation coefficient (R)2) The squares, linear ranges and retention times of (d) are given in table 2. The levels of PC, PE, SM and PI in human plasma were successfully quantified using an external standard method and the average concentrations of all identified phospholipid molecules are listed in table 3.
TABLE 2 phospholipid Standard calibration Curve
TABLE 3 phospholipid molecules identified and plasma concentrations determined thereof
The single pressurized SPE column structure of the present invention is shown in FIGS. 2-5. The working principle is as follows: first using TiO2Filling a column 1 with/KCC-1 core-shell microspheres, and then adding total lipid residues; preparing a pressure plug: the thumb supports against the upper end of the pressure plug, the index finger and the middle finger respectively pull the two handles 401 of the pulling plate 4, so that the hook 402 hooks the hanging lug 602, the telescopic air chamber 3 is stretched to the maximum volume at the moment, and the pressure plug is ready to be completed;
then, the prepared pressure plug is connected with the cylinder 1 in a sealing way through the sealing chute 204 (by rotating), then the knob 601 is rotated to separate the hook 402 from the hanging lug 602, and the telescopic air chamber 3 begins to contract under the action of the extension spring, so that the air pressure in the cylinder 1 rises, and the pressure can be applied to the total lipid residues, and the adsorbent can smoothly pass through.
The single pressurized SPE column has the advantages of simple structure, relatively low cost, convenient operation, portability, small size, no occupation of laboratory space and great contribution to popularization and use in laboratories needing solid phase extraction, and can replace large vacuum equipment.
Claims (10)
1. A method for selectively extracting phospholipids from a plasma sample, comprising the steps of:
a. extracting the total lipid residue from the plasma;
b. 10% by weight of TiO was used2the/KCC-1 core-shell microspheres are used as an adsorbent, and phospholipid in the total lipid residues is adsorbed by using a solid phase extraction method;
c. the phospholipids are eluted from the adsorbent and dried under vacuum to give purified phospholipids.
2. The method for selectively extracting phospholipids from plasma samples according to claim 1, wherein the specific method of the step a is as follows:
mixing 1.5mL of chloroform/methanol solution at a volume ratio of 2:1 per 100. mu.L of plasma sample to obtain a mixture, subjecting the mixture to vortex oscillation for 2 minutes followed by sonication for 15 minutes, then centrifuging at 12000 g at 8 ℃ for 5 minutes to remove insoluble fractions from the mixture, repeating one vortex oscillation, sonication and centrifugation as before, adding 0.2mL of water to the mixture to induce phase separation, and separating to obtain a lower organic phase, and vacuum evaporating the lower organic phase for freeze-drying to obtain a total lipid residue, which is stored at-80 ℃ before further preparation.
3. The method for selectively extracting phospholipids from plasma samples according to claim 1, characterized in that the specific steps of the step b are:
the SPE column is filled with 10 weight percent of TiO2the/KCC-1 core-shell microspheres are activated by methanol and 0.1 percent FA in sequence, and the total lipid residues and 100 mu LFolch solution are passed through an SPE columnAfter loading, the SPE cartridge was washed with chloroform/2-propanol at a 2:1 volume ratio and 10% by mass aqueous methanol to remove fatty acids and all other phosphate group-free lipids.
4. The method of claim 3, wherein the 10 weight percent TiO is selected from the group consisting of2The preparation method of the/KCC-1 core-shell microsphere comprises the following steps:
adding 1.10g of cetylpyridinium chloride and 0.60g per 30mL of water, adding 0.94mL of isopropanol and 30mL of cyclohexane with stirring, then, dropwise adding 3mL of ethyl n-silicate to the mixture using a syringe, vigorously stirring the homogeneous mixture at room temperature for 30 minutes, heating to 70 ℃, and holding for 20 hours, after the reaction, centrifuging the solution at 8000g for 10 minutes, washing the residue three times with acetone, deionized water, and ethanol in this order, then vacuum-drying for 12 hours, and finally, obtaining KCC-1 powder by removing the surfactant from the silica nanoparticles at 500 ℃ in a muffle furnace for 4 hours;
dispersing 2.20g of tetrabutyl titanate in 8.80mL of ethanol, sealing the solution and shaking for 5 minutes under vortex, then adding the mixture to KCC-1 powder under stirring until dry, heating the dried solid material to 100 ℃ for 6 hours, then placing 1g of the heated solid material in an autoclave containing 20mL of water, holding at 60 ℃ for 5 hours, and finally, heat-treating the dried sample in a muffle furnace at 600 ℃ for 5 hours to obtain 10% by weight of TiO2/KCC-1 core-shell microspheres.
5. The method for selectively extracting phospholipids from plasma samples according to claim 1, characterized in that the specific steps of the step c are:
the target phospholipid was eluted from the SPE cartridge using chloroform/methanol at a volume ratio of 1:2 and dried under vacuum, and the resulting phospholipid residue was redissolved in 500. mu.L of Folch solution and filtered through a 0.22. mu.m PTFE membrane before performing the H phospholipid C-MS analysis.
6. The method for selectively extracting phospholipids from plasma samples according to claim 1, characterized in that: the SPE column used in the solid phase extraction method in the step b is a monomer booster-type SPE column, and structurally comprises a column body (1), wherein one end of the column body (1) is provided with a detachably connected pressure plug; the pressure plug comprises a cover body (2) which is hermetically connected with the pipe orifice of the column body (1), the upper end of the cover body (2) is provided with a hollow structure (5), a telescopic air chamber (3) is arranged in the hollow structure (5), and the lower end of the telescopic air chamber (3) is communicated with the column body (1) through an air hole (201) in the cover body (2); the upper end of the telescopic air chamber (3) is provided with a pulling plate (4), and the telescopic air chamber (3) is provided with a tension spring of which two ends are respectively connected with the pulling plate (4) and the cover body (2); the two sides of the hollow structure (5) are provided with sliding grooves (501), and the pull plate (4) is provided with a handle (401) which penetrates through the sliding grooves (501) and protrudes out of the hollow structure (5).
7. The method for selectively extracting phospholipids from plasma samples according to claim 6, characterized in that: the top of the hollow structure (5) is provided with a rotary baffle (6); the upper end of the rotary baffle (6) is provided with a knob (601), and two ends of the rotary baffle are provided with lugs (602); the hollow structure (5) is provided with a transverse limiting groove corresponding to the lug (602), so that the lug (602) can rotate within an angle limited by the transverse limiting groove; the upper end of the handle (401) is provided with a hook (402) corresponding to the lug (602).
8. The method for selectively extracting phospholipids from plasma samples according to claim 6, characterized in that: the pipe orifice of cylinder (1) department is equipped with engaging lug (101), and lid (2) lower extreme is equipped with sealed spout (204), is equipped with breach (202) that correspond with engaging lug (101) on sealed spout (201), is equipped with sealing washer (203) in sealed spout (201).
9. A method for detecting a phospholipid according to any one of claims 1 to 8, comprising: the selective extraction of phospholipids from plasma samples using the method of any one of claims 1 to 8 to obtain purified phospholipids, followed by characterization and quantitative determination using hydrophilic interaction chromatography-mass spectrometry.
10. The phospholipid detection method of claim 9, wherein the hydrophilic interaction chromatography-mass spectrometry is characterized and quantitatively detected by the following specific method:
performing chromatographic separation by using a HILIC chromatographic column at 30 ℃ by using a high performance liquid system, wherein ultrapure water containing 20mM ammonium formate and 0.1% formic acid is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the gradient elution flow rate was 0.6 mL. min-1(ii) a The final optimization conditions are as follows: 0.0 minute, 5% A-95% B; 3.0 min, 5% a-95% B; 4.0 min 12% A-88% B; 12% A-88% B at 10.0 min; 50% A-50% B at 12.0 min; 17.0 min 50% A-50% B; then placing the sample bottle in an autosampler at 8 ℃, injecting 1 mu L of purified phospholipid sample solution into an HPLC system for analysis, and carrying out mass spectrometry by using a triple quadrupole mass spectrometer and the HPLC system; the mass spectrometry apparatus was operated in negative ion mode using electrospray ionization method; the MS full scanning function is used for formal sample analysis, the scanning time is 1s, and the scanning mass range is m/z 600-1000; the sub-ion scanning function is used for tandem mass spectrometry, the optimized collision energy is 22-40V, and the scanning time is set to be 0.1 s; argon was used as the collision gas for MS/MS analysis; other general mass spectral parameters were as follows: capillary voltage, 4.0 KV; cone voltage, 30V; desolventizing temperature is 500 ℃; desolventizing air flow rate, 1000 L.h-1(ii) a Cone gas flow rate, 30 L.h-1。
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