CN113567583A - Method for detecting polar lipid in milk by applying solid phase extraction-supercritical fluid chromatography-mass spectrometry - Google Patents
Method for detecting polar lipid in milk by applying solid phase extraction-supercritical fluid chromatography-mass spectrometry Download PDFInfo
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Abstract
The invention discloses a method for detecting polar lipid in milk by applying solid phase extraction-supercritical fluid chromatography-mass spectrometry, belonging to the field of food detection, and the method comprises the following steps: the method comprises the steps of extracting the total lipid containing polar lipid, enriching and purifying the polar lipid to obtain a polar lipid enrichment substance, and performing supercritical fluid chromatography-mass spectrometry detection analysis after redissolving the polar lipid enrichment substance, so that the enrichment of a sample with extremely low content (less than 5%) of the polar lipid is realized, the operation is simple and convenient, the sample consumption is low, the cost is low, the detection is rapid, the qualitative and quantitative capability is strong, the practicability is strong, the result is stable and reliable, and the method is suitable for uniformly detecting the polar lipid with wide polar range distribution in milk. Can detect 14 kinds of polar lipids within 10 minutes, and is suitable for rapid analysis of polar lipids in various emulsions, dairy products, milk powder and other biological samples.
Description
Technical Field
The invention belongs to the field of food detection, relates to a method for detecting polar lipid in milk, and particularly relates to a method for detecting the polar lipid in the milk by applying solid phase extraction-supercritical fluid chromatography-mass spectrometry.
Background
Milk fat exists in milk in the form of fat globules, and the core triglyceride is surrounded by a membrane composed of polar lipids such as phospholipid and cholesterol, so that the whole emulsion system is kept stable. Cholesterol is an important component of cell membranes, phospholipids in milk are related to the development of the infant brain and intestinal immune system, the protection of gastrointestinal infections and the improvement of memory in the elderly, and Phosphatidylcholine (PC) and Sphingomyelin (SM) are important polar lipids and are structural components required for the rapid growth of organs and cell membranes. About 98% of milk fat is triglycerides, and the content of polar lipids is very low. Thus, in some assays, high levels of triglycerides interfere with the detection of polar lipids. Therefore, it is often necessary to enrich and purify polar lipids. The polar lipid in milk has complex composition and large property difference and is difficult to analyze.
Solid Phase Extraction (SPE) is one of the effective methods of sample preparation, with the basic principle similar to liquid-liquid extraction, which is capable of adsorbing and enriching and purifying analytes from sample solutions on solid adsorbent packing. For the enrichment and purification of polar lipids, the existing patents and documents aim at the enrichment of phospholipids, but most of the existing methods have the problem of sphingomyelin and cholesterol loss. Compared with phospholipid, cholesterol has polarity closer to that of triglyceride, so that the separation and purification difficulty is increased. In addition, patent CN106153763A discloses separation and purification of relatively polar lipids from liquid, but this method has problems of complicated operation, easy loss of cholesterol and sphingomyelin, and large milk fat consumption in a single sample.
In the related art, few reports have reported that the composition of cholesterol, phospholipid and sphingolipid in milk can be simultaneously, qualitatively and quantitatively detected, and various different detection methods are often needed, so that data cannot be unified, the milk fat consumption is increased, and the detection of some rare dairy products is limited. In addition, the chromatographic detection method of polar lipids in the related art is generally long in time, so that the molecular species of the polar lipids are difficult to realize accurate qualitative determination.
Disclosure of Invention
Therefore, in order to overcome the defects in the prior art, the first object of the present invention is to provide a method for detecting polar lipids in milk by solid phase extraction-supercritical fluid chromatography-mass spectrometry, which comprises the following steps:
the method comprises the steps of extracting the total lipid containing polar lipid, enriching and purifying the polar lipid to obtain a polar lipid enrichment substance, and performing supercritical fluid chromatography-mass spectrometry detection analysis after redissolving the polar lipid enrichment substance. The detection method can realize the unified determination of cholesterol, phospholipid and sphingolipid, optimizes the solid phase extraction conditions to ensure that the cholesterol and sphingolipid can be retained in polar lipid eluent, and realizes the rapid qualitative and quantitative detection.
The second purpose of the invention is to provide an application of the method for detecting polar lipid in milk by using solid phase extraction-supercritical fluid chromatography-mass spectrometry, which is used for detecting components of milk liquid raw materials and milk products.
Further, the step of extracting the total lipid material containing polar lipids comprises:
adding an organic solvent A into the emulsion raw material, shaking and mixing uniformly, adding an organic solvent B, shaking and mixing uniformly, carrying out ultrasound, carrying out ice-water bath ultrasound for 5-20 minutes, adding a solution C, shaking and mixing uniformly to promote layering, and thus obtaining a mixture I;
centrifuging the obtained mixture I by a centrifuge, transferring a lower-layer organic solution, adding an organic solvent D into the upper-layer solution for secondary extraction and purification, centrifuging by the centrifuge again, transferring the lower-layer organic solution, combining the organic solutions extracted twice, washing by a solution C, shaking, uniformly mixing, centrifuging by the centrifuge again, removing the upper-layer solution, and drying the lower-layer solution by nitrogen to obtain a total lipid extract containing polar lipids;
wherein the organic solvent A is methanol, and the volume ratio of the emulsion raw material to the organic solvent A is 1: 10-6: 10;
the organic solvent B is chloroform, and the volume ratio of the emulsion raw material to the organic solvent B is 1: 20-6: 20;
the solution C is a sodium chloride solution, the mass percentage concentration of the sodium chloride solution is 0.5-20%, the volume ratio of the emulsion raw material to the solution C for promoting layering is 1: 1-1: 2, and the volume ratio of the emulsion raw material to the solution C for washing is 9: 1-18: 1;
the organic solvent D is a mixed solution of chloroform and methanol, and the weight ratio of chloroform: the volume ratio of the methanol is as follows: 12: 1-1: 1; the volume ratio of the emulsion raw material to the organic solvent D is 1: 10-6: 10.
The best extraction effect of the total lipid substance containing polar lipid is realized by selecting the organic solvent A, the organic solvent B, the solution C and the organic solvent D and selecting and adjusting the range of each proportion under the condition of each proportion.
Further, the step of enriching and purifying the polar lipids to obtain a polar lipid concentrate comprises:
firstly, activating a solid-phase extraction column, wherein the filler of the solid-phase extraction column is 1g/6mL of silica gel, and n-hexane is used for activation;
dissolving 30-200 mg of the total lipid extract containing polar lipids in an organic solvent E to obtain a sample liquid, and loading the sample liquid on an activated solid-phase extraction column;
eluting the column with organic solvent F and organic solvent G sequentially to obtain neutral lipid eluate containing triglyceride;
eluting the column with organic solvent H, organic solvent I and organic solvent J sequentially to obtain eluent rich in polar lipid, and blow-drying with nitrogen to obtain polar lipid concentrate
Wherein the organic solvent E is chloroform: mixed solution of methanol according to the volume ratio of 1: 1-8: 1; the mass-volume ratio of the total lipid extract containing polar lipid to the organic solvent E is 30mg:1 mL-100 mg:1 mL;
the organic solvent F is n-hexane: mixed solution of ethyl ether according to the volume ratio of 100: 1-5: 1; the mass-volume ratio of the total lipid extract containing polar lipids to the organic solvent F is 5mg:1 mL-25 mg:1 mL;
the organic solvent G is n-hexane: mixed solution of ethyl ether according to the volume ratio of 10: 1-2: 1; the mass-volume ratio of the total lipid extract containing polar lipids to the organic solvent G is 10mg:1 mL-50 mg:1 mL;
the organic solvent H is n-hexane: mixed solution of ethyl ether according to the volume ratio of 3: 1-0.5: 1; the mass-volume ratio of the total lipid extract containing polar lipids to the organic solvent H is 30mg:1 mL-90 mg:1 mL;
the organic solvent I is methanol, and the mass-volume ratio of the total lipid extract containing polar lipid to the organic solvent I is 5mg:1 mL-25 mg:1 mL;
the organic solvent J is chloroform: methanol: water is mixed solution according to the volume ratio of 3:5: 2; the mass-to-volume ratio of the total lipid extract containing polar lipids to the organic solvent J is 10mg:1 mL-50 mg:1 mL.
Through the selection of the organic solvent E, the organic solvent F, the solution G, the organic solvent H and the organic solvent I and the selection and adjustment of the range of the mixture ratio, the enrichment and purification of the polar lipid containing the polar lipid are realized with less sample consumption under the conditions of the mixture ratio, and the enrichment of 14 large polar lipids can be realized.
Further, the conditions for performing the supercritical fluid chromatography-mass spectrometry detection analysis on the polar lipid-enriched substance are as follows:
supercritical fluid chromatography adopts Waters Acquity ultra-efficient phase-matching chromatograph UPC2The chromatographic column is Acquity UPC2BEH, 150 mm. times.3.0 mm, 1.7 μm, Waters, USA; the column temperature is 55 ℃, the back pressure is 1500psi, and the sample injection volume is 2 mu L; eluent A is supercritical CO2The purity is more than or equal to 99.99 percent, the eluent B is methanol/water solution containing ammonium formate, the volume percentage of the methanol is 90 to 100 percent, the content of the ammonium formate is 5 to 35mM, the elution gradient is 0min, and the concentration is 5 percent B; 5.5min, 48% B; 10min, 48% B;
the mass spectrum adopts a Waters synapse Q-TOF-MS mass spectrometer, the flow rate of collision gas (argon) is 50L/h, the flow rate of desolvation gas (nitrogen) is 700L/h, the temperature of an ion source is 100 ℃, the desolvation temperature is 400 ℃, the voltage of a taper hole is 30eV, the low-energy collision is 6eV, the high-energy collision is 20-45eV, and the mass-to-charge ratio scanning range is 100-1600 m/z.
In one embodiment, the ultrasonic conditions are 40 kHz; the centrifugation was carried out at 25 ℃ and 8000rpm for 10 min.
In one embodiment, the flow rate of the solid phase extraction elution is 0.03-0.1 mL/min, preferably 0.05 mL/min.
In one embodiment, the reconstitution solvent used for the reconstitution of the polar lipid concentrate is chloroform: the mass volume ratio of the polar lipid enrichment to the redissolution solvent is 1.5mg:1 mL-10 mg:1mL, preferably 5mg:1mL, of the mixed solution of methanol at the volume ratio of 2: 1.
Further, the polar lipid comprises: cholesterol, ceramide Cer, phosphatidylglycerol PG, phosphatidylethanolamine PE, phosphatidylinositol PI, lysophosphatidylethanolamine LPE, phosphatidic acid PA, monosialoganglioside GM3Phosphatidylserine PS, phosphatidylcholine PC, bis-sialylganglioside GD3Sphingomyelin SM, lysophosphatidylcholine LPC and cardiolipin CL.
The invention has the beneficial effects that:
the detection method comprises the following steps: the method comprises the steps of extracting the total lipid containing polar lipid, enriching and purifying the polar lipid to obtain a polar lipid enrichment substance, and performing supercritical fluid chromatography-mass spectrometry detection analysis after redissolving the polar lipid enrichment substance, so that the enrichment of a sample with extremely low content (less than 5%) of the polar lipid is realized, the operation is simple and convenient, the sample consumption is low, the cost is low, the detection is rapid, the qualitative and quantitative capability is strong, the practicability is strong, the result is stable and reliable, and the method is suitable for uniformly detecting the polar lipid with wide polar range distribution in milk. Can detect 14 kinds of polar lipids within 10 minutes, and is suitable for rapid analysis of polar lipids in various emulsions, dairy products, milk powder and other biological samples.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a graph of the results of thin layer chromatography of each eluent enriched and purified with polar lipids of example 1, under the thin layer chromatography conditions: 20 × 20cm silica gel glass plate, after spotting in developing solvent n-hexane: diethyl ether: acetic acid (80:20:1, v/v/v) was allowed to develop, the plate was removed when the solvent front developed to 1cm from the plate front, dried and developed with iodine, as follows.
FIG. 2 is a graph showing the results of thin layer chromatography of each eluent for enrichment and purification of polar lipids in example 3;
FIG. 3 is a graph showing the results of thin layer chromatography of each eluent for enrichment and purification of polar lipids in example 5;
FIG. 4 is a total ion flux chromatogram of supercritical fluid chromatography of example 1;
FIG. 5 is a total ion flux chromatogram of supercritical fluid chromatography of example 2;
FIG. 6 is a supercritical fluid chromatography total ion flux chromatogram of example 3;
FIG. 7 is a total ion flux chromatogram of supercritical fluid chromatography of example 4;
FIG. 8 is a total ion flux chromatogram of supercritical fluid chromatography in example 5.
Detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto: hereinafter, specific embodiments of the present invention will be described in detail so that those skilled in the art can more clearly understand how to practice the present invention. It should be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that these embodiments are intended to illustrate and not limit the scope of the invention.
The polar lipids of the present invention comprise the following 14 major classes: cholesterol, ceramide (Cer), Phosphatidylglycerol (PG), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI), Lysophosphatidylethanolamine (LPE), Phosphatidic Acid (PA), monosialoganglioside (GM)3) Phosphatidylserine (PS), Phosphatidylcholine (PC), bis-sialylganglioside (GD)3) Sphingomyelin (SM), Lysophosphatidylcholine (LPC), and Cardiolipin (CL).
The emulsion raw material in the invention refers to white or yellowish opaque liquid secreted by mammals such as human, cattle, sheep, donkey or camel and the like from mammary glands; the milk product is liquid, powder, gel, paste, solid, concentrate, suspension or instant food prepared from cow milk, sheep milk, donkey milk or camel milk and processed products thereof as main raw materials, with or without appropriate amount of vitamins, minerals and other adjuvants.
Materials and reagents used in the present invention:
phosphatidylglycerol PG (16:0/18:1), phosphatidylethanol PE (14:0/14:0), phosphatidylinositol PI (16:1/16:0), lysophosphatidylethanolamine LPE (18:1/0:0), phosphatidic acid PA
(16:0/18:1), monosialoganglioside GM3Sphingomyelin SM (d18:1/23:0), lysophosphatidylcholine LPC (18:1/0:0) and bisialoganglioside GD3The standards were purchased from Larodan Fine Chemicals AB, Sweden.
Ceramide Cer (d18:1/16:0), cardiolipin CL (14:0/14:0/14:0/14:0) standard: avanti Polar Lipids, usa; phosphatidylserine PS (14:0/14:0) standard: sigma, USA; cholesterol standard: china Addin Inc.; silica gel solid phase extraction column 1g/6 mL: shanghai' an spectral laboratory science and technology company; chromatographic grade n-hexane: china tetam technologies; chromatographic grade methanol: tedia high Purity solutions Inc., USA; chromatographic grade chloroform: china national drug reagents Inc.; high-purity carbon dioxide (purity is more than or equal to 99.999%): wuxin Sn Instrument gas Co.
Supercritical fluid chromatography conditions in the examples: the chromatographic column is Acquity UPC2BEH (150 mm. times.3.0 mm, 1.7 μm, Waters, USA); the column temperature is 55 ℃, the back pressure is 1500psi, and the sample injection volume is 2 mu L; eluent a is supercritical CO2(the purity is more than or equal to 99.99 percent), the eluent b is methanol/water solution (the volume percentage of methanol is 97 percent), the eluent b contains 20mM ammonium formate, the flow rate of the mobile phase is 1.1mL/min, the elution gradient is 0min, and the content of the eluent b is 5 percent; 5.5min, 48% eluent b; 10min, 48% eluent b.
Mass spectrometry conditions in the examples: the flow rate of collision gas (argon) is 50L/h, the flow rate of desolvation gas (nitrogen) is 700L/h, the ion source temperature is 100 ℃, the desolvation temperature is 400 ℃, the taper hole voltage is 30eV, the low-energy collision is 6eV, the high-energy collision is 20-45eV, and the mass-to-charge ratio scanning range is 100-1600 m/z.
The invention is further illustrated by the following examples.
Example 1: detection of polar lipids in breast milk samples
1) Extraction of total lipid material containing polar lipids:
adding 10mL of methanol into 4mL of breast milk, namely the emulsion raw material, shaking for 30 seconds, mixing uniformly, adding 20mL of chloroform, shaking for 15 minutes, mixing uniformly, carrying out ultrasonic ice-water bath for 10 minutes, adding 7.5mL of sodium chloride solution with the mass percentage concentration of 0.9%, promoting layering, shaking for 60 seconds, and mixing uniformly to obtain a mixture I.
Centrifuging the obtained mixture I by a centrifuge under the following conditions: centrifuge at 8000rpm for 10min at 25 deg.C, remove the lower organic solution, and add 15mL of a solution to the upper layer as chloroform: performing secondary extraction and purification on mixed solution of methanol with the volume ratio of 6:1, shaking, uniformly mixing, centrifuging by a centrifuge, transferring lower-layer organic solution, combining the organic solution extracted twice, washing by 0.75mL of sodium chloride solution with the mass percentage concentration of 0.9%, shaking, uniformly mixing, centrifuging by the centrifuge, discarding upper-layer solution, and drying the lower-layer solution by using nitrogen to obtain the total lipid extract containing polar lipids.
2) Enriching and purifying the polar lipid to obtain a polar lipid enrichment:
firstly, activating a solid-phase extraction column, wherein the filler of the solid-phase extraction column is 1g/6mL of silica gel, and the activation is realized by using normal hexane; 50mg of the total lipid extract containing polar lipids was dissolved in 1mL of chloroform: mixing methanol with a volume ratio of 4:1 to obtain a sample solution, and loading the sample solution on an activated solid-phase extraction column;
5mL of n-hexane were used successively: mixed solution of ethyl ether and 3mL of n-hexane according to the volume ratio of 50: 1: eluting the column with diethyl ether at a volume ratio of 6:1 to obtain neutral lipid eluate containing most triglyceride;
1mL of n-hexane was used successively: diethyl ether in a volume ratio of 1:1 mixed solution, 6mL of methanol and 3mL of chloroform: methanol: eluting the column with water according to a mixed solution with a volume ratio of 3:5:2 to obtain an eluent rich in polar lipid, and drying the eluent with nitrogen to obtain a polar lipid enrichment substance.
3) Performing supercritical fluid chromatography-mass spectrometry detection analysis on the polar lipid enrichment:
cholesterol, ceramide (Cer), Phosphatidylglycerol (PG), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI), Lysophosphatidylethanolamine (LPE), Phosphatidic Acid (PA), Phosphatidylserine (PS), Phosphatidylcholine (PC), Sphingomyelin (SM) and Lysophosphatidylcholine (LPC) standards are prepared into a stock solution of polar lipid mixed standard solution by using chloroform methanol (2:1, v/v). Quantitative analysis is realized by adopting an external standard method through a concentration-peak area standard curve, and quantitative analysis is carried out by using a Lipidplast and Progenetics MetScope self-built local database in Progenetics QI software.
The results are as follows:
1. the thin layer chromatography results, see fig. 1, show that most of the triglycerides are effectively removed and cholesterol is retained in the components of the polar lipid concentrate.
2. Taking 3 parts of 10mg breast milk polar lipid enrichment as a blank sample, and performing mass transfer on the blank sample by using chloroform: redissolving the methanol into 5mg/mL according to a mixed solution with the volume ratio of 2:1, respectively adding mixed standard solutions with the high, medium and low concentration levels, wherein the content of the mixed standard solutions is about 1.2 times, 1 time and 0.8 time of the content of each polar lipid in the breast milk polar lipid enrichment, carrying out methodology verification, carrying out 3-time paralleling under each standard adding level, and calculating the intra-day precision, the inter-day precision and the recovery rate of the method by combining with a polar lipid standard curve, and referring to the table 1 and the table 2.
TABLE 1 Linear regression equation for cholesterol, ceramide and phospholipid standards
TABLE 2 precision and recovery of cholesterol, ceramide and phospholipid standards
3. The results of the supercritical fluid chromatography are shown in FIG. 4. Mass spectrum detection results: 187 polar lipids, among which 1 cholesterol, 19 ceramides (Cer), 9 Phosphatidylglycerols (PG), 31 Phosphatidylethanolamines (PE), 12 Phosphatidylinositols (PI), 9 Lysophosphatidylethanolamines (LPE), 20 Phosphatidic Acids (PA), 17 Phosphatidylserines (PS), 28 Phosphatidylcholines (PC), 22 Sphingomyelins (SM), 8 Cardiolipins (CL), and 11 Lysophosphatidylcholines (LPC), were detected in breast milk. Sphingomyelin (SM) is the most abundant phospholipid.
Example 2: detection of gangliosides in breast milk samples
1) Extracting ganglioside enrichment:
4mL of deionized water was added to 4mL of breast milk, followed by shaking for 30 seconds and mixing, followed by addition of 21.6mL of methanol and 10.8mL of chloroform. After gentle shaking at 150rpm for 30 minutes at 25 ℃, the resulting mixture was centrifuged at 8000rpm for 10 minutes at 25 ℃ using a centrifuge. The supernatant rich in polar lipids was collected and 4mL of deionized water and 2mL of chloroform were added to the lower pellet: and carrying out secondary extraction on the methanol by using a mixed solution with the volume ratio of 1: 2. After gentle shaking at 150rpm for 30 minutes at 25 ℃, the mixture was centrifuged at 8000rpm for 10 minutes at 25 ℃ using a centrifuge. The polar lipid-rich supernatant was collected, and the polar lipid-rich supernatants from the two extractions were combined and added to 10.4mL of deionized water, followed by gentle inversion 4 times and centrifugation at 8000rpm for 10 minutes at 25 ℃ using a centrifuge. Collecting the ganglioside-enriched supernatant. To the remaining mixture was added 4mL of a potassium chloride solution with a concentration of 0.01mol/L and centrifuged at 8000rpm for 10 minutes at 25 ℃ with a centrifuge, and the ganglioside-rich supernatant was collected. And combining the supernatants which are extracted twice and are rich in ganglioside, and drying the supernatants by using nitrogen to obtain ganglioside enrichment.
2) Carrying out supercritical fluid chromatography-mass spectrometry detection analysis on the obtained ganglioside enrichment:
monosialoganglioside (GM)3) And bis-sialylganglioside (GD)3) The standard substance is prepared from chloroform: preparing mixed solution of methanol according to the volume ratio of 2:1 to prepare stock solution of ganglioside mixed standard solution. Quantitative analysis is realized by adopting an external standard method through a concentration-peak area standard curve, and quantitative analysis is carried out by using a Lipidplast and Progenetics MetScope self-built local database in Progenetics QI software.
The results are as follows:
1. taking 3 ganglioside concentrates enriched from 4mL breast milk as blank samples, adding 0.5mL chloroform: redissolving with methanol mixed solution at volume ratio of 2:1, adding mixed standard solutions with high, medium and low concentration levels of 1.2 times, 1 time and 0.8 time of each ganglioside content in the breast milk ganglioside concentrate, performing methodology verification, performing 3 parallels at each standard addition level, and calculating day-to-day precision, day-to-day precision and recovery rate of the method, see tables 3 and 4.
TABLE 3 Linear regression equation for gangliosides
TABLE 4 precision and recovery of ganglioside standards
2. The results of the supercritical fluid chromatography are shown in FIG. 5. Mass spectrum detection results: a total of 27 gangliosides, among them monosialoganglioside (GM) was detected in breast milk3)16, bis-sialylganglioside (GD)3)11 kinds of the Chinese herbal medicines. Bis-sialoganglioside (GD) in breast milk3) Concentration of Monosialoganglioside (GM)3) 3.5 times of the total weight of the powder.
Example 3: detection of polar lipids in camel milk samples
1) Extraction of total lipid material containing polar lipids:
adding 10mL of methanol into 4mL of camel milk, shaking for 30 seconds, mixing uniformly, adding 20mL of chloroform, shaking for 15 minutes, mixing uniformly, performing ultrasonic treatment in an ice-water bath for 10 minutes, adding 7.5mL of sodium chloride solution with the mass percentage concentration of 0.9%, promoting layering, shaking for 60 seconds, and mixing uniformly to obtain a mixture I.
The obtained mixture I is centrifuged by a centrifuge under the following conditions: centrifuge at 8000rpm for 10 minutes at 25 ℃, remove the lower organic solution and add 15mL of chloroform: and (3) carrying out secondary extraction and purification on the mixed solution with the volume ratio of methanol of 6:1, shaking, uniformly mixing, centrifuging by using a centrifuge, transferring the lower-layer organic solution, combining the organic solutions extracted twice, washing by using 0.75mL of sodium chloride solution with the mass percentage concentration of 0.9%, shaking, uniformly mixing, centrifuging by using the centrifuge, discarding the upper-layer solution, and drying the lower-layer solution by using nitrogen to obtain the total lipid extract containing polar lipids.
2) Enriching and purifying the polar lipid to obtain a polar lipid enrichment:
firstly, activating a solid-phase extraction column, wherein the filler of the solid-phase extraction column is 1g/6mL of silica gel, and the activation is realized by using normal hexane; 55mg of the total lipid extract containing polar lipids was dissolved in 1mL of chloroform: mixing methanol with a volume ratio of 4:1 to obtain a sample solution, and loading the sample solution on an activated solid-phase extraction column;
5mL of n-hexane were used successively: mixed solution of ethyl ether and 3mL of n-hexane according to the volume ratio of 50: 1: eluting the column with diethyl ether at a volume ratio of 4:1 to obtain neutral lipid eluate containing most of triglyceride;
2mL of n-hexane were used successively: diethyl ether in a volume ratio of 1:1 mixed solution, 4mL of methanol and 3mL of chloroform: methanol: eluting the column with water according to a mixed solution with a volume ratio of 3:5:2 to obtain an eluent rich in polar lipid, and drying the eluent with nitrogen to obtain a polar lipid enrichment substance.
3) Performing supercritical fluid chromatography-mass spectrometry detection analysis on the polar lipid enrichment:
cholesterol, ceramide (Cer), Phosphatidylglycerol (PG), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI), Lysophosphatidylethanolamine (LPE), Phosphatidic Acid (PA), Phosphatidylserine (PS), Phosphatidylcholine (PC), Sphingomyelin (SM) and Lysophosphatidylcholine (LPC) standards are prepared into a stock solution of polar lipid mixed standard solution by using chloroform methanol (2:1, v/v). Quantitative analysis is realized by adopting an external standard method through a concentration-peak area standard curve, and quantitative analysis is carried out by using a Lipidplast and Progenetics MetScope self-built local database in Progenetics QI software.
The results are as follows:
1. the results of thin layer chromatography, see figure 2, show that most of the triglycerides are effectively removed, but that at the same time cholesterol is lost in part with the elution of neutral lipid.
2. The same as in example 1.
3. The results of the supercritical fluid chromatography are shown in FIG. 6. Mass spectrum detection results: 170 polar lipids were detected in camel milk, including 1 cholesterol, 19 ceramides (Cer), 7 Phosphatidylglycerols (PG), 33 Phosphatidylethanolamines (PE), 10 Phosphatidylinositols (PI), 1 Lysophosphatidylethanolamines (LPE), 16 Phosphatidic Acids (PA), 16 Phosphatidylserines (PS), 27 Phosphatidylcholines (PC), 22 Sphingomyelins (SM), 7 Cardiolipins (CL), and 11 Lysophosphatidylcholines (LPC). Phosphatidylethanolamine (PE) is the most abundant phospholipid.
Example 4: detection of polar lipids in milk fat globule membrane samples
1) Lipid extraction:
dissolving 1g of milk fat globule membrane powder in 4.5mL of deionized water, adding 10mL of methanol, shaking for 30 seconds, mixing uniformly, adding 20mL of chloroform, shaking for 2 minutes, mixing uniformly, extracting for 10 minutes in an ultrasonic ice-water bath, adding 6mL of sodium chloride solution with the mass percentage concentration of 0.9% after extraction is finished, promoting layering, shaking for 60 seconds, and mixing uniformly to obtain a mixture I. The resulting mixture I was centrifuged at 8000rpm for 10 minutes at 25 ℃ using a centrifuge, the lower organic solution was removed, and 15mL of a chloroform: performing secondary extraction and purification on mixed solution with the volume ratio of methanol of 6:1, shaking, uniformly mixing, centrifuging by a centrifuge, transferring lower-layer organic solution, combining the organic solution extracted twice, washing by 0.75mL of sodium chloride solution with the mass percentage concentration of 0.9%, shaking, uniformly mixing, centrifuging by the centrifuge, discarding upper-layer solution, and drying the lower-layer solution by using nitrogen to obtain the total lipid extract containing polar lipid, wherein the content of triglyceride in a milk fat globule membrane is less, so that the total lipid extract can be used as a polar lipid concentrate.
2) And (3) carrying out supercritical fluid chromatography-mass spectrometry detection analysis on the obtained polar lipid enrichment:
cholesterol, ceramide (Cer), Phosphatidylglycerol (PG), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI), Lysophosphatidylethanolamine (LPE), Phosphatidic Acid (PA), Phosphatidylserine (PS), Phosphatidylcholine (PC), Sphingomyelin (SM) and Lysophosphatidylcholine (LPC) standards are prepared into a stock solution of polar lipid mixed standard solution by using chloroform methanol (2:1, v/v). Quantitative analysis is realized by adopting an external standard method through a concentration-peak area standard curve, and quantitative analysis is carried out by using a Lipidplast and Progenetics MetScope self-built local database in Progenetics QI software.
The results are as follows:
1. the same as in example 1.
2. The same as in example 1.
3. The results of the supercritical fluid chromatography are shown in FIG. 7. Mass spectrum detection results: the milk fat globule membrane detects 146 polar lipids in total, including 1 cholesterol, 19 ceramides (Cer), 5 Phosphatidylglycerols (PG), 20 Phosphatidylethanolamines (PE), 11 Phosphatidylinositols (PI), 1 Lysophosphatidylethanolamines (LPE), 14 Phosphatidic Acids (PA), 12 Phosphatidylserines (PS), 28 Phosphatidylcholines (PC), 22 Sphingomyelin (SM), 4 Cardiolipins (CL), and 9 Lysophosphatidylcholines (LPC). Phosphatidylethanolamine (PE) is the most abundant phospholipid.
Example 5: detection of polar lipids in formula milk samples
1) Lipid extraction:
dissolving 1g of formula milk powder in 4mL of deionized water, adding 10mL of methanol, shaking for 30 seconds, mixing uniformly, adding 20mL of chloroform, shaking for 5 minutes, mixing uniformly, extracting for 10 minutes in an ultrasonic ice-water bath, adding 8mL of sodium chloride solution with the mass percentage concentration of 1.0% after extraction is finished, promoting layering, shaking for 60 seconds, and mixing uniformly to obtain a mixture I.
The resulting mixture I was centrifuged at 8000rpm for 10 minutes at 25 ℃ using a centrifuge, the lower organic solution was removed, and 15mL of a chloroform: and (3) carrying out secondary extraction and purification on the mixed solution with the volume ratio of methanol of 6:1, shaking, uniformly mixing, centrifuging by using a centrifuge, transferring the lower-layer organic solution, combining the organic solutions extracted twice, washing by using 0.75mL of sodium chloride solution with the mass percentage concentration of 0.9%, shaking, uniformly mixing, centrifuging by using the centrifuge, removing the upper-layer solution, and drying the lower-layer solution by using nitrogen to obtain the total lipid extract containing polar lipids.
2) Enrichment and purification:
firstly, activating a solid-phase extraction column, wherein the filler of the solid-phase extraction column is 1g/6mL of silica gel, and the activation is realized by using normal hexane; 50mg of the total lipid extract containing polar lipids was dissolved in 1mL of chloroform: mixing methanol with a volume ratio of 4:1 to obtain a sample solution, and loading the sample solution on an activated solid-phase extraction column;
5mL of n-hexane were used successively: mixed solution of ethyl ether and 3mL of n-hexane according to the volume ratio of 50: 1: eluting the column with diethyl ether at a volume ratio of 8:1 to obtain neutral lipid eluate containing most of triglyceride;
1mL of n-hexane was used successively: diethyl ether in a volume ratio of 1:1 mixed solution, 4mL of methanol and 3mL of chloroform: methanol: eluting the column with water according to a mixed solution with a volume ratio of 3:5:2 to obtain an eluent rich in polar lipid, and drying the eluent with nitrogen to obtain a polar lipid enrichment substance.
3) Performing supercritical fluid chromatography-mass spectrometry detection analysis on the polar lipid enrichment:
cholesterol, ceramide (Cer), Phosphatidylglycerol (PG), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI), Lysophosphatidylethanolamine (LPE), Phosphatidic Acid (PA), Phosphatidylserine (PS), Phosphatidylcholine (PC), Sphingomyelin (SM) and Lysophosphatidylcholine (LPC) standards are prepared into a stock solution of polar lipid mixed standard solution by using chloroform methanol (2:1, v/v). Quantitative analysis is realized by adopting an external standard method through a concentration-peak area standard curve, and quantitative analysis is carried out by using a Lipidplast and Progenetics MetScope self-built local database in Progenetics QI software.
The results are as follows:
1. the results of thin layer chromatography, see figure 3, show that cholesterol is retained in the polar lipid fraction, but that triglycerides are retained more in the polar lipid eluate.
2. Same as example 1
3. The results of the supercritical fluid chromatography are shown in FIG. 8. Mass spectrum detection results: the formula milk detects 207 polar lipids in total, wherein cholesterol is 1, ceramide (Cer) is 19, Phosphatidylglycerol (PG) is 9, Phosphatidylethanolamine (PE) is 35, Phosphatidylinositol (PI) is 15, and lysophosphatidylethanolamine
8 kinds of (LPE), 28 kinds of Phosphatidic Acid (PA), 19 kinds of Phosphatidylserine (PS), 28 kinds of Phosphatidylcholine (PC), 22 kinds of Sphingomyelin (SM), 7 kinds of Cardiolipin (CL), and 16 kinds of Lysophosphatidylcholine (LPC). Phosphatidylcholine (PC) is the most abundant phospholipid.
According to the preferred embodiments, the detection method disclosed by the invention can uniformly detect 14 major polar lipids with wide polar range distribution in milk within 10 minutes in all the preferred embodiments, and has the advantages of rapidness in detection, strong qualitative and quantitative capability, strong practicability, stable and reliable result and suitability. Comparative example 2, example 3 the neutral lipid removing effect in the enrichment and purification was similar to that of example 2, but a small amount of cholesterol was lost; comparative example 2, example 5 enrichment and clarification cholesterol retention was similar to example 2, but more triglyceride was retained. However, in general, the solid phase extraction method for enrichment and purification in the present invention can be applied to the enrichment of samples with very low content of polar lipids (less than 5%), and has the advantages of simple operation, low sample consumption and low cost.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (9)
1. A method for detecting polar lipid in milk by using solid phase extraction-supercritical fluid chromatography-mass spectrometry is characterized by comprising the following steps: the method comprises the steps of extracting the total lipid containing polar lipid, enriching and purifying the polar lipid to obtain a polar lipid enrichment substance, and performing supercritical fluid chromatography-mass spectrometry detection analysis after redissolving the polar lipid enrichment substance.
2. The method for detecting polar lipids in milk by solid phase extraction-supercritical fluid chromatography-mass spectrometry as claimed in claim 1, wherein said step of extracting the total lipid material containing polar lipids comprises:
adding an organic solvent A into the emulsion raw material, shaking and mixing uniformly, adding an organic solvent B, shaking and mixing uniformly, performing ice-water bath ultrasound for 5-20 minutes, adding a solution C, shaking and mixing uniformly to promote layering to obtain a mixture I;
centrifuging the obtained mixture I by a centrifuge, transferring a lower-layer organic solution, adding an organic solvent D into the upper-layer solution for secondary extraction and purification, centrifuging by the centrifuge again, transferring the lower-layer organic solution, combining the organic solutions extracted twice, washing by a solution C, shaking, uniformly mixing, centrifuging by the centrifuge again, removing the upper-layer solution, and drying the lower-layer solution by nitrogen to obtain a total lipid extract containing polar lipids;
wherein the organic solvent A is methanol, and the volume ratio of the emulsion raw material to the organic solvent A is 1: 10-6: 10;
the organic solvent B is chloroform, and the volume ratio of the emulsion raw material to the organic solvent B is 1: 20-6: 20;
the solution C is a sodium chloride solution, the mass percentage concentration of the sodium chloride solution is 0.5-20%, the volume ratio of the emulsion raw material to the solution C for promoting layering is 1: 1-1: 2, and the volume ratio of the emulsion raw material to the solution C for washing is 9: 1-18: 1;
the organic solvent D is a mixed solution of chloroform and methanol, and the weight ratio of chloroform: the volume ratio of the methanol is as follows: 12: 1-1: 1; the volume ratio of the emulsion raw material to the organic solvent D is 1: 10-6: 10.
3. The method for detecting polar lipids in milk by using solid phase extraction-supercritical fluid chromatography-mass spectrometry as claimed in claim 1, wherein said step of enriching and purifying polar lipids to obtain a polar lipid enrichment comprises:
firstly, activating a solid-phase extraction column, wherein the filler of the solid-phase extraction column is 1g/6mL of silica gel, and n-hexane is used for activation;
dissolving the total lipid extract containing polar lipids in an organic solvent E to obtain a sample solution, and loading the sample solution onto an activated solid-phase extraction column;
eluting the column with organic solvent F and organic solvent G sequentially to obtain neutral lipid eluate containing triglyceride;
eluting the column with organic solvent H, organic solvent I and organic solvent J sequentially to obtain eluent rich in polar lipid, and blow-drying with nitrogen to obtain polar lipid concentrate
Wherein the organic solvent E is chloroform: the methanol is mixed liquid according to the volume ratio of 1: 1-8: 1; the mass-volume ratio of the total lipid extract containing polar lipid to the organic solvent E is 30mg:1 mL-100 mg:1 mL;
the organic solvent F is n-hexane: ether (A)
Mixed liquid according to the volume ratio of 100: 1-5: 1; the mass-volume ratio of the total lipid extract containing polar lipids to the organic solvent F is 5mg:1 mL-25 mg:1 mL;
the organic solvent G is n-hexane: mixed solution of ethyl ether according to the volume ratio of 10: 1-2: 1; the mass-volume ratio of the total lipid extract containing polar lipids to the organic solvent G is 10mg:1 mL-50 mg:1 mL;
the organic solvent H is n-hexane: mixed solution of ethyl ether according to the volume ratio of 3: 1-0.5: 1; the mass-volume ratio of the total lipid extract containing polar lipids to the organic solvent H is 30mg:1 mL-90 mg:1 mL;
the organic solvent I is methanol, and the mass-volume ratio of the total lipid extract containing polar lipid to the organic solvent I is 5mg:1 mL-25 mg:1 mL;
the organic solvent J is chloroform: methanol: water is mixed solution according to the volume ratio of 3:5: 2; the mass-to-volume ratio of the total lipid extract containing polar lipids to the organic solvent J is 10mg:1 mL-50 mg:1 mL.
4. The method for detecting polar lipid in milk by using solid phase extraction-supercritical fluid chromatography-mass spectrometry as claimed in claim 1, wherein the conditions for performing supercritical fluid chromatography-mass spectrometry detection analysis on the enriched polar lipid are as follows:
supercritical fluid chromatography adopts Waters Acquity ultra-efficient phase-matching chromatograph UPC2The chromatographic column is Acquity UPC2BEH，150mm×3.0mm, 1.7 μm, Waters, USA; the column temperature is 55 ℃, the back pressure is 1500psi, and the sample injection volume is 2 mu L; eluent a is supercritical CO2The eluent b is methanol/water solution containing ammonium formate, the volume percentage of the methanol is 90-100%, the content of the ammonium formate is 5-35 mM, the elution gradient is 0min, and the concentration is 5% b; 5.5min, 48% b; 10min, 48% b;
the mass spectrum adopts a Waters synapse Q-TOF-MS mass spectrometer, the flow rate of collision gas is 50L/h, the flow rate of desolvation gas is 700L/h, the temperature of an ion source is 100 ℃, the temperature of desolvation is 400 ℃, the voltage of a taper hole is 30eV, low-energy collision is 6eV, high-energy collision is 20-45eV, and the mass-to-charge ratio scanning range is 100-1600 m/z.
5. The method for detecting polar lipids in milk by solid phase extraction-supercritical fluid chromatography-mass spectrometry as claimed in claim 2, wherein said ultrasonic conditions are 40 kHz; the centrifugation was carried out at 25 ℃ and 8000rpm for 10 min.
6. The method for detecting polar lipids in milk by using solid phase extraction-supercritical fluid chromatography-mass spectrometry as claimed in claim 3, wherein the flow rate of the solid phase extraction elution is 0.03-0.1 mL/min, preferably 0.05 mL/min.
7. The method for detecting polar lipids in milk by using solid phase extraction-supercritical fluid chromatography-mass spectrometry as claimed in claim 1, wherein the redissolving solvent for redissolving the enriched polar lipids is chloroform: the mass volume ratio of the polar lipid enrichment to the redissolution solvent is 1.5mg:1 mL-10 mg:1mL, preferably 5mg:1mL, of the mixed solution of methanol at the volume ratio of 2: 1.
8. The method for detecting polar lipids in milk using solid phase extraction-supercritical fluid chromatography-mass spectrometry according to claim 1, wherein said polar lipids comprise: cholesterol, ceramide Cer, phosphatidylglycerol PG, phosphatidylethanolamine PE, phosphatidylinositol PI, lysophosphatidylethanolamine LPE, phosphatidic acid PA, monosialoganglioside GM3Phosphorus, phosphorusPhosphatidylserine PS, phosphatidylcholine PC, bis-sialylganglioside GD3Sphingomyelin SM, lysophosphatidylcholine LPC and cardiolipin CL.
9. An application of a method for detecting polar fat in milk by applying solid phase extraction-supercritical fluid chromatography-mass spectrometry is characterized in that the method is used for detecting components of milk liquid raw materials and milk products.
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