WO2020232813A1 - 一种食品脂质提取及检测食品脂质的方法 - Google Patents

一种食品脂质提取及检测食品脂质的方法 Download PDF

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WO2020232813A1
WO2020232813A1 PCT/CN2019/095969 CN2019095969W WO2020232813A1 WO 2020232813 A1 WO2020232813 A1 WO 2020232813A1 CN 2019095969 W CN2019095969 W CN 2019095969W WO 2020232813 A1 WO2020232813 A1 WO 2020232813A1
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dichloromethane
methanol
phospholipids
glycerides
lipids
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French (fr)
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陈历俊
赵军英
姜铁民
乔为仓
刘茜
刘妍
张明辉
王筱迪
张滢
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Beijing Sanyuan Foods Co Ltd
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Beijing Sanyuan Foods Co Ltd
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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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4055—Concentrating samples by solubility techniques
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4055—Concentrating samples by solubility techniques
    • G01N2001/4061—Solvent extraction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G01N2001/4083—Concentrating samples by other techniques involving separation of suspended solids sedimentation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/04—Dairy products

Definitions

  • the invention relates to the field of food extraction and detection, in particular to a method for food lipid extraction and food lipid detection.
  • Lipids are the main nutrients in food, mainly including 8 categories: fatty acids, glycerides (including triglycerides, diglycerides, monoglycerides), glycerophospholipids (phosphatidylcholine PC, phosphatidylethanolamine PE, phospholipids Acylinositol PI, phosphatidylserine PS, phosphatidylglycerol PG, phosphatidic acid PA), sphingolipids (ceramide Cer, sphingomyelin SM), glycolipids (ganglioside GM, GD), sterol lipids, Pregnenolone lipids, polyvinyls. Since the concept of lipidomics was proposed in 2003, the importance of food lipidomics for health and disease prevention has gradually been recognized.
  • Breast milk lipids are the second largest component of human breast milk after lactose, mainly including glycerides, glycerophospholipids, sterol lipids and glycolipids, and the content in colostrum is as high as 40g/L.
  • Breast milk not only provides 40-55% of the energy for the growth and development of breastfed infants, phospholipids are also an indispensable component of the membrane structure of cells and organelles, and have the function of resisting various metabolic diseases.
  • Gangliosides are brain lipids and An important component of the cerebral cortex.
  • the breastfeeding rate in my country is less than 29%, and the strong support of breastmilk composition data can be one of the effective ways to promote breastfeeding; breastmilk composition is also the gold standard for infant formula design.
  • breast milk lipids are affected by maternal genes, diet, environment and other factors, to clarify the types and contents of breast milk lipids, it is necessary to test representative breast milk samples from different regions, stages of lactation, and different races to establish a breast milk lipid database.
  • breast milk is the only food for infants and young children, especially breast-fed babies from 0-6 months, so it is more difficult to collect breast milk samples, and the sample size collected is small. Therefore, the establishment of the breast milk lipid database relies on high-throughput detection methods, which not only increase the detection throughput, but also reduce the amount of breast milk samples, and ensure the safety of the inspectors during the detection of large quantities of samples. Therefore, the establishment of a high-throughput, low sample consumption, and safe breast milk lipid detection method is a necessary prerequisite for analyzing breast milk components to promote breastfeeding and develop more nutritious and healthy infant formula powder.
  • lipid composition is an important aspect of their nutritional functions.
  • it is necessary to detect the lipid composition of existing infant formula foods, and conduct qualitative and quantitative analysis of the lipid composition in different infant food ingredients. Both require high-throughput and safe detection technology.
  • the detection methods for lipids in foods, especially breast milk, dairy products, and ingredients are mainly based on the detection of glycerides, phospholipids (including glycerophospholipids and sphingomyelin), fatty acids, and gangliosides.
  • the samples are subjected to different methods. After processing, use different instrument methods to detect.
  • the pretreatment methods mainly include the traditional Folch method and Bligh&Dyer method to extract glycerides or phospholipids:
  • the specific process of solid phase extraction includes: 50mg lipid extract is dissolved in 1mL chloroform/methanol (95:5, v/v) mixture, silica gel column (500mg) is first used with 5mL chloroform/methanol (95:5, v/v) ) Activation, and then adding lipid solution, triglyceride was eluted with 10mL chloroform/methanol (95:5, v/v), phospholipid was eluted with 5mL methanol and 5mL chloroform/methanol/water (3:5:2, v/v) /v) The mixed solution is eluted. After the eluate is evaporated to dryness, it is reconstituted in chloroform/methanol (2:1, v/v) for detection.
  • This method has not been used for breast milk lipid extraction, and is mainly used for the extraction of animal milk lipids.
  • Food, especially breast milk and infant formula powders and ingredients for lipid testing equipment and methods are mainly as follows: Gas chromatography-Mass spectrometry (GC-MS) to detect fatty acid composition; liquid chromatography (LC) or Supercritical Fluid Chromatography (SFC) combined with Quadrupole-Time of Flight-Mass Spectrometer (Q-TOF-MS) to detect triglycerides and their fatty acid composition; liquid-phase evaporation photodetector , Thin layer chromatography analysis of phospholipids or LC-Q-TOF-MS to detect phospholipids and their fatty acid composition; tandem mass spectrometry or LC-Q-TOF-MS to detect gangliosides.
  • the various detection methods are as follows:
  • the main parameters of mass spectrometry are as follows: positive ion mode, scanning range m/z50-2000, flow rate of nitrogen for desolvation is 800L/h, collision gas is argon, ion
  • the source temperature is 120°C
  • the solvent removal temperature is 450°C
  • the capillary voltage is 3.0kV
  • the sampling cone voltage is 25V
  • the sampling cone gas flow rate is 20L/h
  • the low collision energy is 4eV
  • the high gradient collision energy is 20-60eV.
  • liquid chromatography fluorescence detectors are often used to detect phospholipids, but this method cannot determine the composition of fatty acids in phospholipids. Or use the MS/MS ALL mode of Triple-TOF-MS to analyze phospholipids.
  • the detection conditions are as follows: collision energy 45eV in positive and negative ion mode, voltage 0.96kV, back pressure 1.25psi.
  • liquid phase parameters Hilic column, 150 ⁇ 4.6mm ID, 2.7 ⁇ m dp
  • the elution gradient is 0-10min, 40% B; 15min, 45% B ; 25min, 80%B; 40min, 90%B.
  • the flow rate is 0.7mL/min, the injection volume is 5 ⁇ L; the mass spectrometry parameters are positive ion mode and negative ion mode, curve desolvent tube temperature 200°C, ion source temperature 200°C, N2 spray gas flow rate 1.5L/min, scanning range m /z 200-1200, ion accumulation time 100ms; secondary scan: scanning range m/z200-1000, ion accumulation time 30ms, parent ion separation parameters: width 3, time 20ms, m/z600-1000, collision dissociation parameters: Energy 100%, collision gas 100%, time 50ms.
  • Mass spectrometry conditions ion spray voltage 4000V, ion source temperature 400°C, declustering voltage 40V, ion source gas 1 is 40psi, ion source gas 2 is 35psi, curtain gas 15psi, collision energy 40V.
  • the shotgun method was proposed in 2004 to directly enter the extracted lipids into the mass spectrometer for ionization and detection at the same time.
  • the analysis speed is fast, but in order to avoid the saturation of the detector and contaminate the mass spectrometer, the sample needs to be diluted about 200 times and subject to high abundance or easy ionization.
  • the ion inhibition of lipids makes it difficult to qualitatively or quantitatively analyze low-concentration lipids.
  • LC-Q-TOF-MS can be used to detect triglycerides and their fatty acid composition, phospholipids and their fatty acid composition, and gangliosides in foods, especially breast milk, maternal and infant milk, and ingredients
  • the existing methods use In the detection of a large number of samples, the following shortcomings exist in the establishment of a database of breast milk and lipids of different raw materials:
  • Glycerides and phospholipids can be processed once through a sample, and then the non-polar glycerides and polar glycerophospholipids can be separated by a solid phase extraction column and then detected separately. However, gangliosides need to be sampled for pre-processing.
  • the highly toxic substance chloroform is used. When exposed to light, chloroform will react with oxygen in the air and gradually decompose to generate highly toxic phosgene, and has an anesthetic effect, which is harmful to the heart, liver and kidney. The process of testing large quantities of samples will cause greater harm to human health. .
  • the shotgun method can be used to detect phospholipids and glycerides at the same time, on the one hand, the sample needs to be diluted 200 times, which makes it difficult to detect some low-concentration lipids.
  • gangliosides need to be analyzed by LC-Q-TOF-MS.
  • the commonly used methods are the detection of phospholipids by high performance liquid chromatography with fluorescence detector, the detection of glycerides and gangliosides by liquid chromatography mass spectrometry, and the detection of fatty acid composition in samples by gas chromatography mass spectrometry. Therefore, at least two sets of tandem mass spectrometry and liquid mass spectrometry are required to analyze food lipids.
  • High-performance liquid-mass spectrometry is currently the main technology for detecting milk-derived lipids at the molecular level.
  • the research summary of food lipid detection based on this technology is shown in Table 1. It mainly lacks high-throughput simultaneous qualitative and quantitative analysis of phospholipids and glycerol. Complete detection technology for esters. There is only one report on the simultaneous qualitative analysis of phospholipids and glycerides, which did not detect gangliosides, and did not achieve quantitative analysis.
  • the current technology mostly qualitatively and quantify phospholipids and glycerides at the molecular level. Different samples are required to extract lipids and then tested separately. This results in a large amount of sample usage and multiple pre-processing operations, which is time-consuming and consuming. Power, material consumption, and phospholipids are mostly quantified at the subclass level through the use of evaporative light detectors, that is, only the content of phosphatidylcholine, etc. can be quantified, and the content of phosphatidylcholine of different fatty acid chains cannot be determined.
  • the present invention aims to establish a method for low-sample, safe and high-throughput sequencing based on the characteristics of food lipids and the principle of LC-Q-TOF-MS for detecting food lipids, that is, using a small amount of samples and passing safer lipids
  • qualitative and quantitative detections are performed simultaneously or separately.
  • the establishment of a food, especially breast milk lipid database provides technical support for the design of scientific food lipids, especially mother and infant food lipids.
  • the invention provides a method for extracting food lipids and detecting food lipids.
  • the present invention provides a method for extracting food lipids, which can extract lipids in foods, including fatty acids, glycerides, phospholipids, and gangliosides.
  • the phospholipids include Glyerophospholipids and sphingomyelin.
  • the method includes mixing a food sample with a mixed internal standard, then mixing it with a lipid extraction solvent and ultrapure water multiple times and centrifuging multiple times, combining multiple centrifugation and the upper phase obtained contains ganglioside, and combining multiple centrifugation
  • the lower organic phase obtained later contains phospholipids, fatty acids and glycerides.
  • the lipid extraction solvent is dichloromethane or a mixture of dichloromethane, methanol and ultrapure water.
  • the food sample may be solid food or liquid food that can be eaten directly, or solid food that needs to be reconstituted into a liquid state for consumption.
  • the solid food or liquid food can be directly used for lipid extraction.
  • the solid food sample is a solid food that needs to be reconstituted into a liquid and then eaten, the solid food needs to be reconstituted into a liquid according to the method of repacking food, and then lipid extraction is performed.
  • the amount of food samples used can be only 50-350 ⁇ L of liquid food samples or liquids prepared according to the method of distributing food preparations or 0.05-0.1 g powder solid food samples. It is possible to extract lipids from a very small amount of food samples, which provides technical support for subsequent lipid detection and content determination, and has the advantages of wide applicability and small sample consumption.
  • the mixed internal standard is specifically one or more of deuterated glycerides, different types of deuterated phospholipids, and different types of deuterated sphingolipids.
  • the different types of deuterated phospholipids and different types of deuterated sphingolipids mean that their types are different from the types of phospholipids and sphingolipids in food samples.
  • the mixed internal standard may include one or more of the following: glycerides, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, sphingomyelin, neuroleptic Amide etc.
  • the mixed internal standard can eliminate the shortcomings that different experimenters and different batches of samples are difficult to analyze uniformly due to differences in operation and instrument status, and has the advantages of improving data comparability and accuracy.
  • the principle of determining the amount of the mixed internal standard is: the mass concentration of each lipid after the mixed internal standard is added is 60-140% of the mass concentration of the same type of lipid in the original sample, preferably 80%-120%, more preferably Specifically, the mass concentration of each lipid after the mixed internal standard is added is equivalent to the mass concentration of the same type of lipid in the original sample; that is, the test result of the food sample is used to determine the content of each lipid through the external standard curve, and the concentration of each lipid in the mixed internal standard
  • the addition amount is 60-140% of the content of each lipid determined by the external standard curve; preferably 80%-120%; more preferably 100%.
  • the present invention uses dichloromethane to replace the chloroform used in the lipid extraction, separation and purification process to improve the safety of the method.
  • Dichloromethane is the least toxic of methane chloride, and its toxicity is only 0.11% of that of carbon tetrachloride. . At the same time, it has the advantages of being miscible with lipids and can be used for lipid extraction, separation and purification.
  • the food lipid extraction method of the present invention includes the following steps: using a mixture of dichloromethane and methanol as a lipid extraction solvent for the first centrifugation to obtain an upper phase and a lower organic phase.
  • the volume ratio of methylene chloride, methanol and ultrapure water is 1-3.5:1-3:0.2-0.55, preferably 1-3:1-3:0.2-0.55, More preferably: 9:10:2.
  • the amount of dichloromethane is 0.5-2mL/100 ⁇ L liquid food sample or 1-3mL/0.05g powder solid food sample; preferably 0.6-1.2mL/100 ⁇ L liquid food sample or 1.5-2.5 mL/0.05g powder solid food sample. Under this amount, lipids can be fully extracted, and it has the advantage of separating different lipids.
  • the ultrapure water and dichloromethane are added in two portions, and the methanol is added in one time.
  • the two addition amounts of the ultrapure water and methylene chloride are the same.
  • the two addition amounts of the ultrapure water and dichloromethane are different.
  • the ratio of the two added amounts of the ultrapure water is 0.15-0.4:0.1-0.3; more preferably 0.2:0.25. In an embodiment of the present invention, the ratio of the two added amounts of dichloromethane is 0.8-1.0:0.85-1.1; more preferably 0.9:1.0. In one embodiment of the present invention, after adding ultrapure water, dichloromethane and methanol, shake well and sonicate, and then add the remaining ultrapure water and dichloromethane. In one embodiment of the present invention, the ultrasonic treatment is performed under the following conditions: 20-50KHz, 600-900W, temperature 20-30°C, ultrasonic for 1-10 minutes; preferably 40KHz, 800W, 25°C, ultrasonic 5 minutes.
  • the food lipid extraction method further includes the following steps: taking the upper phase obtained by the first centrifugation, adding dichloromethane to it to perform the second centrifugation to obtain the upper phase and the lower organic phase. phase.
  • the amount of dichloromethane used is 0.2-1.8 mL/100 ⁇ L liquid food sample or 1-3 mL/0.05 g powder solid food sample.
  • the amount of dichloromethane is 0.6-1.2 mL/100 ⁇ L liquid food sample or 1.5-2.5 mL/0.05 g powder solid food sample.
  • the food lipid extraction method further includes the following steps: taking the lower organic phase obtained from the first centrifugation and adding ultrapure water, methanol and dichloromethane to it to perform the third centrifugation , The upper phase and the lower organic phase are obtained.
  • the volume ratio of the ultrapure water, methanol and dichloromethane is 0.3-1:1-3.5:0.5-3, preferably: 0.3-0.6:1-2:0.5-1 .
  • the amount of dichloromethane used is 0.3-1 mL/100 ⁇ L liquid food sample or 0.3-1 mL/0.05 g powder solid food sample.
  • the food lipid extraction method further includes the following steps: combining the upper phase obtained by the second centrifugation with the upper phase obtained by the third centrifugation, and drying with nitrogen to obtain ganglion Glycosides.
  • the present invention separates the gangliosides into the upper phase and separates them from fatty acids/glycerides/phospholipids by limiting the lipid extraction solvent, and can obtain an extract containing only gangliosides, which has the advantage of efficient separation .
  • the food lipid extraction method further includes a step of purifying the ganglioside.
  • the step of purifying the ganglioside is specifically: dissolving the obtained ganglioside in a 60-90% methanol solution, then centrifuging, taking the upper phase, and drying with nitrogen. , That is, the ganglioside is obtained.
  • the amount of the methanol solution is 100-350 ⁇ L/100 ⁇ L liquid food sample or 100-350 ⁇ L/0.05 g powder solid food sample.
  • the food lipid extraction method further includes the following steps: combining the lower organic phase obtained by the second centrifugation with the lower organic phase obtained by the third centrifugation, and drying with nitrogen to obtain Phospholipids, fatty acids and glycerides.
  • the phospholipids, fatty acids and glycerides obtained by the invention do not contain insoluble magazines such as protein precipitation, and have the advantage of protecting the liquid quality system.
  • the food lipid extraction method further includes the step of purifying the phospholipids, fatty acids and glycerides.
  • the step of purifying the phospholipids, fatty acids and glycerides is specifically: dissolving the obtained phospholipids, fatty acids and glycerides in a dichloromethane and methanol solution of ammonium acetate, and then centrifugation to take The lower organic phase is dried with nitrogen to obtain the phospholipids, fatty acids and glycerides.
  • the ratio of dichloromethane to methanol in the dichloromethane and methanol solution of ammonium acetate is 0.6-1.5:0.6-1.2; preferably 1:1.
  • the concentration of the ammonium acetate is 5-15 mmol/L, preferably 8-12 mmol/L, more preferably 10 mmol/L.
  • the amount of the dichloromethane and methanol solution of ammonium acetate is 0.2-2.5 mL/100 ⁇ L liquid food sample or 0.5-2.5 mL/0.05 g powder solid food sample.
  • the lipid extraction solvent is dichloromethane:methanol with a specific volume ratio, and the purpose is to separate the gangliosides into the upper phase; the lower organic phase and the upper phase obtained are separated Separation:
  • the organic phase uses a specific volume ratio of dichloromethane:methanol:water to extract the gangliosides again, and the aqueous phase uses dichloromethane to extract the fatty acids, glycerides and phospholipids again.
  • the food lipid extraction method further comprises the following steps: dissolving the obtained phospholipids, fatty acids and glycerides in a volume ratio of 0.6-1.5:0.6-1.2 (preferably 2:1) Dichloromethane and methanol solutions are used to obtain a reconstituted lipid solution, and then the reconstituted lipid solution is separated by a silica gel column to obtain the glycerides/fatty acids and the phospholipids, respectively.
  • the amount of the dichloromethane and methanol solution is 0.2-2.5 mL/100 ⁇ L liquid food sample or 0.5-2.5 mL/0.05 g powder solid food sample.
  • the silica gel column separation specifically includes the following steps:
  • Activated silica gel column Use 2-5mL n-hexane to activate silica gel column (6mL volume, 1g adsorbent) Silica gel bonded cartridges (Dikma ProElut Si, 6-mL volume, 1g sorbents), discard the eluent, and then re Pour the lipid solution into the column;
  • n-hexane/diethyl ether 8:2, V/V solution to the column, collect the eluent, and then add 2-5 mL of n-hexane/diethyl ether (1: 1. V/V) solution, collect the eluate; after drying with nitrogen, the glycerides/fatty acids are obtained;
  • Elute phospholipids add 2-5mL methanol to the column, then add 1-3.5mL methanol and 1-3.5mL dichloromethane/methanol/water (3:5:2, V/V/V), collect the elution After drying with nitrogen, the phospholipid is obtained.
  • the method of the present invention simultaneously extracts fatty acids, glycerides, phospholipids and gangliosides in foods, can simultaneously achieve high-efficiency extraction and separation of lipids, and has the advantages of small sample consumption, high extraction efficiency and separation of different lipids .
  • the present invention provides a method for extracting food lipids, which can extract fatty acids/glycerides/phospholipids and gangliosides respectively, and the method includes the following steps:
  • the present invention provides a method for extracting food lipids, which can extract fatty acids/glycerides, phospholipids, and gangliosides respectively, and the method includes the following steps:
  • the present invention also provides a method for detecting food lipids; it can specifically detect phospholipids/glycerides/fatty acids and gangliosides.
  • the method for detecting food lipids includes the following steps:
  • Detection of phospholipids/glycerides/fatty acids take the lipids or glycerides/fatty acids and phospholipids extracted in the above-mentioned food lipid extraction method and dilute them to between the upper and lower limits of lipid quantitative detection, using LC -Q-TOF-MS(AB Sciex 6600) Detect under the following conditions: Chromatographic column: Kinetex C18 Column (150mm ⁇ 4.6mm, 2.6 ⁇ m; Phenomenex, Torrance, CA, USA) or Kinetex C18 Column (50mm ⁇ 3mm, 2.6 ⁇ m; Phenomenex, Torrance, CA, USA); column temperature is 30-50°C; mobile phase A is 5nmol/L ammonium acetate solution (methanol ⁇ acetonitrile ⁇ water, 1 ⁇ 1 ⁇ 1, V ⁇ V ⁇ V), mobile phase B is 5nmol/L ammonium acetate solution (isopropanol), flow rate 0.3-0.8mL/min, injection volume 1-3 ⁇ L.
  • the detection object is the lipid extracted in the above-mentioned food lipid extraction method
  • the detection of the phospholipid/glyceride/fatty acid is as follows: Perform the elution gradient as follows:
  • the method for detecting food lipids performs an elution gradient according to the following table :
  • a switch valve can be used behind the liquid chromatography column. After a certain retention time, when the phospholipid peak is completed, the triglycerides eluted in the liquid chromatography column are discharged. To the waste tank, so as not to enter the mass spectrometer due to high ion concentration and contaminate the mass spectrometer.
  • Mass spectrometry is detected separately in positive ion mode and negative ion mode.
  • the precursor ion detection parameters accumulation time 0.15secs, scan range (tof masses) 100-1300Da, elution time (duration) 15min, heating Gas (GS1) 50-65psi, auxiliary heating gas (GS2) 50-65psi, curtain gas (CUR) 30-45psi, ion source temperature (TEM) 500-650°C, ion spray voltage (ISVF) 4500-5500V (if negative ion Mode, only change this parameter to -4500V), declustering voltage (DP) 80V, and collision energy (CE) 10V.
  • TEM ion source temperature
  • ISVF ion spray voltage
  • DP declustering voltage
  • CE collision energy
  • Mass spectrometry secondary debris detection parameters accumulation time (accumulation time) 0.05secs, scanning range (tof masses) 50-1300Da, heating gas (GS1) 50-65psi, auxiliary heating gas (GS2) 50-65psi, curtain gas (CUR) 30-45psi, ion source temperature (TEM) 500-650°C, ion spray voltage (ISVF) 4500-5500V (if negative ion mode, only change this parameter to -450V), collision energy (CE) 30-50V, collision energy Superposition (CES) 10-25V.
  • the method for detecting food lipids includes the following steps:
  • gangliosides take the gangliosides extracted in the above-mentioned food lipid extraction method and dilute them to the upper limit and lower limit of the quantitative detection of lipids, using LC-Q-TOF-MS ( AB Sciex 6600) Detect under the following conditions: Chromatographic column: BEH C18 (100 ⁇ 2.1mm, 1.7um; Waters), column temperature is 30-50°C; mobile phase A is 90ml water+10ml methanol+0.1ml 1mol/L Ammonium acetate, mobile phase B is 100ml methanol + 0.1ml 1mol/L ammonium acetate, the flow rate is 0.3mL/min, and the injection volume is 10 ⁇ L.
  • the gangliosides are detected according to the following table with an elution gradient:
  • Mass spectrometry mode is negative ion mode, precursor ion detection parameters: accumulation time (accumulation time) 0.25secs, scanning range (tof masses) 100-2000Da, elution time (duration) 14min, heating gas (GS1) 35-50psi, auxiliary heating gas (GS2) 30-45psi, curtain air (CUR) 10-25psi, ion source temperature (TEM) 300-450°C, ion spray voltage (ISVF) -3500-4500V, declustering voltage (DP) -30-50V, collision Energy (CE) -35-50V.
  • Mass spectrometry secondary debris detection parameters accumulation time (accumulation time) 0.05secs, scanning range (tof masses) 50-2000Da, heating gas (GS1) 35-50psi, auxiliary heating gas (GS2) 30-45psi, curtain gas (CUR) 10-25psi, ion source temperature (TEM) 300-450°C, ion spray voltage (ISVF) -3500-4500V, declustering voltage (DP) -35-50V, collision energy (CE) -35-45V, -collision energy Superposition (CES) 15-30V, ion release delay (IRD) 30, ion release width (IRW) 15.
  • the present invention also provides a lipid qualitative analysis method, wherein the qualitative analysis uses Peakview2.2 software.
  • the lipid qualitative analysis method includes the following:
  • glycerides are +NH 4 + positive ions in the positive ion mode. According to the glyceride database that comes with this software, qualitative analysis is based on the accurate m/z value of the precursor ion first, and then based on the two The grade fragment (diglyceride) information determines the fatty acid composition. If the fatty acid identified by the fragments can combine the number of carbon atoms and double bonds in the parent ion glyceride, then the glyceride is qualitative;
  • phospholipid types have different qualitative methods, among which PA, PG, PI, PS, PE can be imported in negative ion mode according to Peakview's own database, and then set the summation ion to -H after, PC plus ions and CH 3 COO -, according to the first parent ion qualitative analysis, and then determining whether the fatty acid composition is consistent with the corresponding number of carbon atoms in the parent ion and a double bond in accordance with debris, can be qualitatively consistent if this phospholipid.
  • SM and Cer are analyzed in the positive ion mode. When the SM plus ion is H + , 184 secondary characteristic fragments are used for qualitative analysis, and Cer is qualitatively analyzed by 264 secondary characteristic fragments;
  • gangliosides After importing the corresponding data according to the ganglioside database of Peakview in the negative ion mode, the gangliosides are first searched according to the exact molecular weight, isotope and molecular formula, and the retrieved gangliosides Lipid then determines whether it contains a specific ganglioside according to whether there is sialic acid in the secondary fragments (the ratio of mass to nucleus is 290.1).
  • the present invention also provides a lipid quantitative analysis method, wherein the quantitative analysis adopts MultieQuant 3.0.2 software for quantitative analysis of different lipids.
  • the lipid quantitative analysis method includes the following:
  • the internal standard method is mainly used for the quantification of different glycerides.
  • the internal standard contains a deuterated internal standard mixture of 9 kinds of glycerides. According to the content of the corresponding glyceride in the measured dairy product, the content of the internal standard is similar to the content of the corresponding glyceride in the dairy product. Other glycerides choose the deuterated internal standard with similar retention time to calculate the content.
  • the external standard method is used to establish a standard curve with the area ratio of different representative external standard products and internal standard as the ordinate, and the concentration of different external standards as the abscissa to quantitatively calculate the content of different glycerides in the sample;
  • Phospholipid quantitative analysis Different types of breast milk phospholipid quantitative analysis uses both internal standard method and external standard method.
  • the internal standard method uses Phosphatidylchaline (PC) 31:2 (17:1; 14:1), Phosphatidylserine (PS) 31:2 (17:0-14:1), Alcohol (Phosphatidylinositol, PI) 31:2 (17:0-14:1), Phosphatidylehanolamine (PE) 31:2 (17:0-14:1), Phosphatidylglycerol (PG) 31: 1 (17:0-14:1), Phosphatidic acid (PA), Sphingomyelin (SM) d18:1/17:0, Ceramide (d18:2(4E,8Z)/24 :0)) as an internal standard.
  • PC Phosphatidylchaline
  • PS Phosphatidylserine
  • Alcohol Phosphatidyli
  • the area ratio of the external standard product to the corresponding internal standard is the ordinate
  • the concentration of the external standard is the ordinate to establish a standard curve to calculate the content of different phospholipids in the sample
  • Gangliosides are mainly used to quantify the higher content of monosialoganglioside (GM3) and disialoganglioside (GD3), using external standard method Quantitative. Both GM3 and GD3 standard products contain a variety of monomers composed of different alkyl hydrocarbons and sugars, so the sum of the peak areas with higher content is used as the basis for quantitative calculation. The ganglioside in the sample is quantified, and the sum of different GM3 and GD3 is also calculated. The standard curves of gangliosides GM3 and GD3 are shown in Figure 1 and Figure 2, respectively.
  • the non-polar glycerides, polar phospholipids and gangliosides are eluted and then entered into mass spectrometry detection. It is a single injection of glycerides, phospholipids and The key to gangliosides.
  • the present invention establishes two methods. One method can simultaneously detect glycerides, fatty acids, phospholipids and gangliosides, which can be used for the detection of extracted samples containing different types of lipids. It can also detect only glycerides and/or Phospholipids; the other method only detects gangliosides and is used for the detection of extracted ganglioside samples.
  • the glycerides, fatty acids, phospholipids and gangliosides can be extracted through one sampling and processing
  • the literature has methods for extracting gangliosides alone, and only a few literatures extract phospholipids, glycerides, and fatty acids together.
  • the present invention can obtain two kinds of ganglioside extract and phospholipid/fatty acid/glyceride extract according to the needs of detection and analysis through one sampling process; and according to the concentration of phospholipid, fatty acid and glyceride in the sample, Improve the accuracy of qualitative analysis and quantitative analysis of phospholipids, fatty acids and glycerides, and reduce the pollution of the instrument.
  • Glycerides, fatty acids and phospholipids can be separated and diluted or concentrated in different multiples for further testing.
  • the present invention uses dichloromethane instead of chloroform to extract lipids, which improves the safety of the experiment.
  • Dichloromethane is the least toxic among methane chlorides, and its toxicity is only 0.11% of that of carbon tetrachloride.
  • one sample needs to use 60-80ml chloroform, and the extraction process needs to be shaken and centrifuged, and the solid phase extraction needs to be eluted with chloroform, and the above process is difficult to complete the closed operation. Therefore, in the process of testing large quantities of samples, not only will it cause health effects on operators and laboratory personnel, but the waste chloroform will cause secondary poisoning during storage, transportation and processing.
  • the present invention uses dichloromethane instead of chloroform in the traditional method.
  • the present invention establishes an LC-Q-TOF-MS method for simultaneously detecting glycerides, phospholipids, ceramides, and sphingolipids, and establishes a separate method suitable for gangliosides Extraction method.
  • This method is suitable for different foods, including liquid and solid, especially breast milk, dairy products and corresponding ingredients.
  • Figure 1 is a standard curve of ganglioside GM3, the abscissa is the concentration and the ordinate is the ionic strength.
  • Figure 2 is: another standard curve of ganglioside GD3, the abscissa is the concentration and the ordinate is the ionic strength.
  • Example 1 Extraction of glycerides, fatty acids, phospholipids and gangliosides from human milk
  • 1#tube 200 ⁇ L breast milk+200 ⁇ L ultrapure water+1.8mL methanol+800 ⁇ L dichloromethane (chromatographically pure) ⁇ manual shake for 10s, ultrasonic for 5min ⁇ add 200 ⁇ L ultrapure water + 800 ⁇ L dichloromethane (chromatographically pure) ⁇ hand Shake well for 10s ⁇ centrifuge, 6000r/min, 15min ⁇ take out the lower organic phase to tube 2# ⁇
  • Activated silica gel column activate the silica gel column with 3mL n-hexane (6mL volume, 1g adsorbent ) Silica gel bonded cartridges (Dikma ProElut Si, 6-mL volume, 1g sorbents) , discard the eluent, and then remove the reconstituted lipid Pour the substance solution into the column;
  • Elute non-polar lipids first add 3 mL of n-hexane/diethyl ether (8:2, V/V) solution to the column, collect the eluent, and then add 3 mL of n-hexane/diethyl ether (1:1, V/V) V) Solution, collect the eluate; after drying with nitrogen, 2mL reconstituted solution is reconstituted, 100 ⁇ L is taken out, 900 ⁇ L reconstituted solution is added, 6000r/min, after 5min centrifugation, the supernatant is taken on the machine to detect glycerides and free fatty acids;
  • Mass spectrometry is detected separately in positive ion mode and negative ion mode.
  • the precursor ion detection parameters accumulation time 0.15secs, scan range (tof masses) 100-1300Da, elution time (duration) 15min, heating Gas (GS1) 60psi, auxiliary heating gas (GS2) 60psi, curtain gas (CUR) 35psi, ion source temperature (TEM) 600°C, ion spray voltage (ISVF) 5500V (if negative ion mode, only change this parameter to -450V ), declustering voltage (DP) 80V, collision energy (CE) 10V.
  • the precursor ion detection parameters accumulation time 0.15secs, scan range (tof masses) 100-1300Da, elution time (duration) 15min, heating Gas (GS1) 60psi, auxiliary heating gas (GS2) 60psi, curtain gas (CUR) 35psi, ion source temperature (TEM) 600°C, ion spray voltage (ISVF) 5500
  • Mass spectrometry secondary debris detection parameters accumulation time (accumulation time) 0.05secs, scanning range (tof masses) 50-1300Da, heating gas (GS1) 60psi, auxiliary heating gas (GS2) 60psi, curtain gas (CUR) 35psi, ion source Temperature (TEM) 600°C, ion spray voltage (ISVF) 5500V (if negative ion mode, only change this parameter to -450V), collision energy (CE) 40V, collision energy superposition (CES) 20V.
  • TAG52:3 (9.57%, 18:2-18:1-16:0)
  • TAG52:2 (7.87%, 18:2-18:0-16:0)
  • TAG54:5 (6.46%, 18:2-18:2-18:1)
  • TAG52:4 (6.25%, 18:2-18:2-16: 0)
  • TAG54:4 (6.18%, 18:1-18:1-18:2)
  • Glyerophospholipids include: 12 types of phosphatidylcholine, 2 types of lysophosphatidylcholine, 24 types of phosphatidylethanolamine, 8 types of lysophosphatidylethanolamine, 7 types of phosphatidylinositol, 1 type of lysophosphatidylinositol, phospholipids 4 kinds of acids, 1 kind of phosphatidylglycerol, 7 kinds of phosphatidylserine.
  • PE36:2 35.45%, 18:0-18:2
  • PS36:2 (10.63%, 18:0-18:2)
  • PE36:1 9.25%, 18: 0-18:1)
  • PC36:2 8.37%, 18:0-18:2)
  • PE36:3 4.20%, 18:1-18:2).
  • Mass spectrometry is detected separately in positive ion mode and negative ion mode.
  • the precursor ion detection parameters accumulation time 0.15secs, scan range (tof masses) 100-1300Da, elution time (duration) 15min, heating Gas (GS1) 55psi, auxiliary heating gas (GS2) 50psi, curtain gas (CUR) 30psi, ion source temperature (TEM) 550°C, ion spray voltage (ISVF) 5000V, declustering voltage (DP) 70V, collision energy (CE) )10V.
  • Mass spectrometry secondary debris detection parameters accumulation time (accumulation time) 0.05secs, scanning range (tof masses) 50-1300Da, heating gas (GS1) 55psi, auxiliary heating gas (GS2) 50psi, curtain gas (CUR) 30psi, ion source Temperature (TEM) 550°C, ion spray voltage (ISVF) 5000V, collision energy (CE) 35V, collision energy superposition (CES) 15V.
  • Example 3 Breast milk glycerides/fatty acids/phospholipids and gangliosides were extracted separately and tested twice
  • Step 1 2# tube + 1.6mL dichloromethane ⁇ centrifugation, 6000r/min, 15min ⁇ take out the upper phase to 3# tube, wait to mix with the upper phase taken out in step 2
  • Step 2 1# tube left organic phase+2mL methanol+0.8mL dichloromethane+1mL ultrapure water ⁇ centrifuged, 6000r/min, 15min ⁇ take out the upper phase and mix with the upper layer of tube 3# in step 1, the upper phase Direct filter loading to detect gangliosides
  • Mass spectrometry is detected separately in positive ion mode and negative ion mode.
  • the precursor ion detection parameters accumulation time 0.15secs, scan range (tof masses) 100-1300Da, elution time (duration) 15min, heating Gas (GS1) 65psi, auxiliary heating gas (GS2) 65psi, curtain gas (CUR) 40psi, ion source temperature (TEM) 650°C, ion spray voltage (ISVF) 5500V, declustering voltage (DP) 70V, collision energy (CE ) 15V.
  • Mass spectrometry secondary debris detection parameters accumulation time (accumulation time) 0.05secs, scanning range (tof masses) 50-1300Da, heating gas (GS1) 65psi, auxiliary heating gas (GS2) 65psi, curtain gas (CUR) 35psi, ion source Temperature (TEM) 600°C, ion spray voltage (ISVF) 5500V (if negative ion mode, only change this parameter to -450V), collision energy (CE) 40V, collision energy superposition (CES) 20V.
  • Step 1 2# tube + 1.6mL dichloromethane ⁇ centrifugation, 6000r/min, 15min ⁇ take out the upper phase to 3# tube, wait to mix with the upper phase taken out in step 2
  • Step 2 1# tube left organic phase+2mL methanol+0.8mL dichloromethane+1mL ultrapure water ⁇ centrifuged, 6000r/min, 15min ⁇ take out the upper phase and mix with the upper layer of tube 3# in step 1, the upper phase Direct filter loading to detect gangliosides
  • Mass spectrometry is detected separately in positive ion mode and negative ion mode.
  • the precursor ion detection parameters accumulation time 0.15secs, scan range (tof masses) 100-1300Da, elution time (duration) 15min, heating Gas (GS1) 65psi, auxiliary heating gas (GS2) 65psi, curtain gas (CUR) 40psi, ion source temperature (TEM) 650°C, ion spray voltage (ISVF) 5500V, declustering voltage (DP) 70V, collision energy (CE ) 15V.
  • Mass spectrometry secondary debris detection parameters accumulation time (accumulation time) 0.05secs, scanning range (tof masses) 50-1300Da, heating gas (GS1) 65psi, auxiliary heating gas (GS2) 65psi, curtain gas (CUR) 35psi, ion source Temperature (TEM) 600°C, ion spray voltage (ISVF) 5500V (if negative ion mode, only change this parameter to -450V), collision energy (CE) 40V, collision energy superposition (CES) 20V.
  • TAG36:0 (8.65%, 4:0-14:0-18:0)
  • TAG38:1 8.00%, 4 :0-16:0-18:1
  • TAG38:0 6.60%, 4:0-16:0-18:0
  • TAG50:1 (4.99%, 18:1-14:0-18:0)
  • TAG40:1 4.95%, 6:0-16:0-18:1)
  • Phospholipids detected 6 kinds of phosphatidyl alcohol, 1 kind of phosphatidylcholine, and the higher contents were: PE36:1 (29.88%, 18:1-18:0), PE36:2 (23.24%, 18:1-18:1), PE36:3 (18.75%, 18:1-18:2)
  • Step 1 2# tube + 1.6mL dichloromethane ⁇ centrifugation, 6000r/min, 15min ⁇ take out the upper phase to 3# tube, wait to mix with the upper phase taken out in step 2
  • Step 2 1# tube left organic phase+2mL methanol+0.8mL dichloromethane+1mL ultrapure water ⁇ centrifuged, 6000r/min, 15min ⁇ take out the upper phase and mix with the upper layer of tube 3# in step 1, the upper phase Direct filter loading to detect gangliosides
  • Mass spectrometry is detected separately in positive ion mode and negative ion mode.
  • the precursor ion detection parameters accumulation time 0.15secs, scan range (tof masses) 100-1300Da, elution time (duration) 15min, heating Gas (GS1) 65psi, auxiliary heating gas (GS2) 65psi, curtain gas (CUR) 40psi, ion source temperature (TEM) 650°C, ion spray voltage (ISVF) 5500V, declustering voltage (DP) 70V, collision energy (CE ) 15V.
  • Mass spectrometry secondary debris detection parameters accumulation time (accumulation time) 0.05secs, scanning range (tof masses) 50-1300Da, heating gas (GS1) 65psi, auxiliary heating gas (GS2) 65psi, curtain gas (CUR) 35psi, ion source Temperature (TEM) 600°C, ion spray voltage (ISVF) 5500V (if negative ion mode, only change this parameter to -450V), collision energy (CE) 40V, collision energy superposition (CES) 20V.
  • TAG52:2 (8.97%, 18:1-16:0-18:1), TAG54:5 (7.61%) , 18:2-18:2-18:1), TAG50:1 (7.41%, 16:0-16:0-18:1), TAG54:6 (7.02%, 18:3-18:1-18 :2), TAG54:3 (6.36%, 18:2-18:2-18:0).
  • PE36:2 (36.49%, 18:1-18:1)
  • PE36:3 (12.64%, 18:1-18:2)
  • PE34:1 (10.44%, 16:0-18:1).
  • this comparative example uses the traditional Folch method for extraction, and the details are as follows:
  • the present invention provides a food lipid extraction method and a lipid detection method.
  • the food lipid extraction method includes mixing a food sample with a mixed internal standard, and then mixing with a lipid extraction solvent and ultrapure water for multiple times. After centrifugation, the upper phase obtained by combining multiple centrifugation contains gangliosides, and the lower organic phase obtained after combining multiple centrifugation contains phospholipids, fatty acids and glycerides.
  • the present invention adopts a small amount of samples, through a relatively safe lipid extraction reagent, after one treatment, after obtaining extracts of different lipids, the qualitative and quantitative detections are performed simultaneously or separately. In order to reduce sample consumption, improve safety, save consumables in the process of sample pretreatment and detection, reduce costs and improve efficiency, it has good economic value and application prospects.

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Abstract

提供了一种食品脂质提取方法和食品中脂质的检测方法。所述提取方法通过将食品样品与超纯水、甲醇和二氯甲烷进行多次混合和离心，多次离心后所得下层有机相即为脂质。该检测方法采用LC-Q-TOF-MS同时或分别对提取得到的脂质进行定性和定量检测，以减少样本用量，提高安全性，节约样品预处理和检测过程中的耗材，降低成本并提高效率。

Description

一种食品脂质提取及检测食品脂质的方法 技术领域
本发明涉及食品提取及检测领域，具体涉及一种食品脂质提取及检测食品脂质的方法。
背景技术
脂质是食品中的主要营养成分，主要包括8大类：脂肪酸、甘油酯(包括甘油三酯、甘油二酯、单甘脂)、甘油磷脂(磷脂酰胆碱PC、磷脂酰乙醇胺PE、磷脂酰肌醇PI、磷脂酰丝氨酸PS、磷脂酰甘油PG、磷脂酸PA)、鞘脂类(神经酰胺Cer、鞘磷脂SM)、糖脂(神经节苷脂GM、GD)、固醇脂类、孕烯醇酮脂、多聚乙烯类。自2003年脂质组学概念被提出以来，食品脂质组学对健康及预防疾病的重要性逐渐被认知。
母乳脂质是人类母乳中仅次于乳糖的第2大组分，主要包括甘油酯、甘油磷脂、固醇脂类和糖脂类，在初乳中含量高达40g/L。母乳不仅为纯母乳喂养婴幼儿生长发育提供了40-55％的能量，磷脂还是细胞和细胞器膜结构不可缺少的成分，并具有抵抗各种代谢疾病的功能，神经节苷脂是大脑脂质和大脑皮层的重要组成物质。我国的母乳喂养率不到29％，母乳成分数据的有力支撑可成为促进母乳喂养的有效方式之一；母乳成分也是婴幼儿奶粉配方设计的黄金标准。因母乳脂质受母亲基因、膳食、环境等因素影响，明确母乳脂质的种类及含量，需检测不同区域、不同哺乳阶段、不同种族等有代表性的母乳样本，建立母乳脂质数据库。但母乳是婴幼儿尤其是0-6月母乳喂养婴儿的唯一食品，所以母乳样本的采集难度较大，且采集的样本量较少。因此，母乳脂质数据库的建立依赖于高通量的检测方法，既要提高检测通量，又要减少母乳样本用量，并确保大批量样本检测过程中检测人员的安全性。所以，建立高通量、低样本消耗、安全的母乳脂质检测方法，是分析母乳成分进而促进母乳喂养并开发更加营养健康的婴幼儿配方粉的必要前提。
各种固体、液体食品设计时，脂质组成是其营养功能的重要方面。尤 其为了设计更加科学合理的婴幼儿配方食品，需对现有的婴幼儿配方食品中脂质组成进行检测，并对不同婴幼儿食品配料中的脂质组成进行定性和定量分析。同样都需要高通量、安全检测技术。
目前，食品尤其是母乳、乳品及配料中脂质的检测方法主要是基于检测甘油酯、磷脂(包括甘油磷脂和鞘磷脂)、脂肪酸、神经节苷脂的不同目标，将样品分别经不同方法前处理后，采用不同的仪器方法检测。其中前处理方法主要包括传统的Folch法和Bligh&Dyer法提取甘油酯或磷脂：
1)Folch法
25倍稀释，2g奶+50g氯仿/甲醇2:1+5mlNaCl(0.05M)，200rpm摇20min，5000rpm离心20min，去除底层氯仿层，上层甲醇层加入35ml氯仿，重复上述过程，下层氯仿层与上次的混合，蒸干，-20℃保存备用；然后用固相萃取柱把中性甘油酯与极性磷脂分开。
固相萃取具体过程包括：50mg脂质提取物溶解在1mL氯仿/甲醇(95:5，v/v)混合液中，硅胶柱(500mg)先用5mL氯仿/甲醇(95:5，v/v)活化，再加入脂质溶液，甘油三酯用10mL氯仿/甲醇(95:5，v/v)洗脱，磷脂用5mL甲醇和5mL氯仿/甲醇/水(3:5:2,v/v/v)混合溶液洗脱，洗脱液蒸干后，复溶到氯仿/甲醇(2:1,v/v)中用于检测。
也可采用以下洗脱方法：脂质：200μL提取的脂质，溶解到1ml氯仿：甲醇(v/v＝2:1)中；活化硅胶柱：用3mL正己烷活化硅胶柱(6mL体积，1g吸附剂)，弃去洗脱液，然后将复溶后的脂质溶液倒入柱子中；洗脱非极性脂：首先向柱子中加入3mL的正己烷/二乙醚(8:2，V/V)溶液，收集洗脱液，然后加入3mL正己烷/二乙醚(1:1，V/V)溶液，收集洗脱液；氮吹干后，复溶待检测；洗脱磷脂：向柱子中加入4mL的甲醇，然后加入2mL甲醇和2mL二氯甲烷/甲醇/水(3:5:2，V/V/V)，收集洗脱液，氮气吹干后，复溶待检测。
2)Bligh&Dyer法
此方法未见有用于母乳脂质提取，主要用于动物乳品脂质的提取。具体如下：500μL奶中加入18.7mL氯仿-甲醇(V/V＝1:2)，然后加入0.62mL氯仿和0.62mL水，旋涡震荡后离心，取出底部有机相，氮气下浓缩，即得到脂质。
3)神经节苷脂的提取方法主要如下：
2ml奶加入8ml甲醇、4ml氯仿、1ml水，超声5min，8800g离心5min；加入2ml水，分层取出上层，剩余物加入6ml甲醇、3ml氯仿、2ml水，超声离心后，取上层与之前的混合。旋转冻干，用1ml1:1的甲醇：水复溶，固相萃取柱富集。
食品尤其是母乳及婴幼儿配方粉和配料中脂质的检测仪器及方法主要如下：气质联用(Gas chromatography-Mass spectrometry，GC-MS)检测脂肪酸组成；液相色谱(liquid chromatography,LC)或超临界流体色谱(Supercritical Fluid Chromatography,SFC)联用四级杆-飞行时间质谱(Quadrupole-Time of Flight-Mass Spectrometer Q-TOF-MS)检测甘油三酯及其脂肪酸组成；液相蒸发光检测器、薄层层析分析磷脂或LC-Q-TOF-MS检测磷脂及其脂肪酸组成；串联质谱或LC-Q-TOF-MS检测神经节苷脂。各种检测方法具体如下：
1)SFC-Q-TOF-MS分析母乳甘油酯时，质谱主要参数如下：正离子模式，扫描范围m/z50-2000，脱溶剂用氮气的流速为800L/h，碰撞气体为氩气，离子源温度为120℃，脱溶剂温度为450℃，毛细管电压为3.0kV，进样锥的电压25V，进样锥气体流速为20L/h，低碰撞能量为4eV，高梯度碰撞能量为20–60eV。
2)目前较多采用液相色谱荧光检测器检测磷脂，但此方法不能确定磷脂中脂肪酸的组成。或采用Triple-TOF-MS的MS/MS  ALL模式分析磷脂，其中检测条件如下：正离子和负离子模式下碰撞能量45eV，电压0.96kV，背压1.25psi。以及LC-MS-IT-TOF分析母乳磷脂的报道，其中液相参数：Hilic色谱柱，150×4.6mm I.D.,2.7μm d.p.，流动相A相为乙腈 -甲酸铵(9:1，v/v；pH＝5.5)，B相为乙腈-甲醇-甲酸铵(55:35:10，v/v/v；pH＝5.5)，洗脱梯度为0–10min，40％B；15min，45％B；25min，80％B；40min，90％B。流速为0.7mL/min，进样体积为5μL；质谱参数为正离子模式和负离子模式下，曲线脱溶剂管温度200℃，离子源温度200℃，N2喷雾气流速1.5L/min，扫描范围m/z 200–1200，离子累积时间100ms；二级扫描：扫描范围m/z200-1000，离子累积时间30ms，母离子分离参数：宽度3，时间20ms，m/z600-1000，碰撞解离参数：能量100％，碰撞气体100％，时间50ms。
3)LC-Q-TOF-MS分析神经节苷脂的参数如下：液相条件：色谱柱BEH C18柱(1.7×150mm，2.1mm i.d.)，柱温50℃，流动相A水/甲醇/乙酸铵(1mmol/L)(90/10/0.1v/v/v)，流动相B甲醇/乙酸铵(1mmol/L)(100/0.1v/v)，洗脱梯度：0min 10％A，0.2min 10％A，8.2min 5％A，12.2min 5％A，12.4min 0％A，18.4min 0％A，18.6min10％A，21min 10％A，流速0.2mL/min。质谱条件：离子喷雾电压4000V，离子源温度400℃，去簇电压40V，离子源气体1为40psi，离子源气体2为35psi，气帘气15psi，碰撞能量40V。
鸟枪法2004年被提出，是将提取脂质直接同时进入质谱离子化并检测，分析速度快，但为避免检测器饱和污染质谱，样品需稀释约200倍，且受高丰度或易离子化脂质的离子抑制作用，致使难以定性或定量分析低浓度脂质。
背景技术的缺陷
尽管LC-Q-TOF-MS可推测同时用于检测食品尤其是母乳、母婴乳品及配料中的甘油三酯及其脂肪酸组成、磷脂及其脂肪酸组成和神经节苷脂，但现有方法用于大量样本的检测，建立母乳和不同原料脂质数据库时存在以下不足：
1)不同脂质需分别提取
甘油酯和磷脂可通过一次样品处理后，采用固相萃取柱将非极性的甘 油酯和极性的甘油磷脂分离后分别检测，但神经节苷脂需另取样品进行前处理。
2)脂质提取需采用高毒物质氯仿
无论是Folch法还是Bligh&Dyer法提取甘油酯和磷脂，还是现有的提取神经节苷脂的方法，均采用高毒物质氯仿。氯仿遇光会与空气中的氧作用，逐渐分解而生成剧毒的光气，且具有麻醉作用，对心、肝、肾有损害，大批量样品检测过程中将对人体健康的产生较大危害。
3)甘油酯、磷脂和神经节苷脂需采用不同的仪器检测
目前尽管可采用鸟枪法同时检测磷脂和甘油酯，但一方面样品需稀释200倍导致部分低浓度的脂质难以被检测到，另一方面神经节苷脂需采用LC-Q-TOF-MS分析。且现有文献报道中，常用的方法是采用高效液相色谱荧光检测器检测磷脂，液质联用检测甘油酯和神经节苷脂，采用气质联用检测样品中的脂肪酸组成。因此，至少需要串联质谱和液质联用两套仪器解析食品脂质。
高效液相-质谱联用是目前在分子水平检测乳源脂质的主要技术，基于此技术的食品脂质检测研究汇总如表1所示，主要缺乏高通量同时定性和定量分析磷脂和甘油酯的成套检测技术。仅有1篇磷脂和甘油酯同时定性分析的报道，并没有检测其中的神经节苷脂，且没有实现定量分析。
表1基于LC-MS技术检测乳源甘油酯和磷脂分子组成的文献汇总
Figure PCTCN2019095969-appb-000001
Figure PCTCN2019095969-appb-000002
综上所述，当前技术多是将磷脂和甘油酯分别在分子水平定性和定量，需不同样品单独提取脂质后分别检测，导致样本使用量大，前处理操作需多次，耗时、耗力、耗材料，并且磷脂多是通过蒸发光检测器，在亚类水平上定量，即仅能定量磷脂酰胆碱等含量，不能确定不同脂肪酸链磷脂酰胆碱等的含量。
发明内容
本发明的目的
本发明旨在基于食品脂质的特性和LC-Q-TOF-MS检测食品脂质的原理，建立样本量少、安全、高通量测序的方法，即采用少量样本，通过较为安全的脂质提取试剂，经过一次处理，得到不同脂质的提取物后，同时或分别对其进行定性和定量检测。以减少样本用量，提高安全性，节约样品预处理和检测过程中的耗材，降低成本并提高效率。以用于定性并定 量分析脂质的组成，建立食品、尤其是母乳脂质数据库，为科学的食品脂质尤其是母婴食品脂质的设计提供技术支持。
本发明的技术方案
本发明提供了一种食品脂质提取及检测食品脂质的方法。
在一些实施方案中，本发明提供了一种食品脂质提取方法，所述食品脂质提取方法可以提取食品中的脂质，包括脂肪酸、甘油酯、磷脂和神经节苷脂，所述磷脂包括甘油磷脂和鞘磷脂。
所述方法包括将食品样品与混合内标混合，然后与脂质提取溶剂和超纯水进行多次混合以及多次离心，合并多次离心后所得上层相包含神经节苷脂，合并多次离心后所得下层有机相包含磷脂、脂肪酸和甘油酯。
优选地，所述脂质提取溶剂为二氯甲烷或二氯甲烷、甲醇和超纯水的混合物。
其中，在所述食品样品可以为可直接食用的固体食品或液体食品、或者需冲调成液态后食用的固体食品。在所述食品样品为可直接食用的固体食品或液体食品的情况下，该固体食品或液体食品可直接用于脂质提取。在所述食品样品为需冲调成液体后食用的固体食品的情况下，需将所述固体食品按分装食品冲调方法冲调成液体，然后进行脂质提取。
在本发明所述食品脂质提取方法中，使用的食品样品的量可以仅为50-350μL液体食品样品或按分装食品冲调方法冲调后的液体或0.05-0.1g粉末固体食品样品。可通过对极少量的食品样品进行脂质的提取，为后续的脂质检测及含量的测定提供了技术支撑，具有适用性广、样本用量少的优点。
其中，所述混合内标具体为：氘代甘油酯、不同种类氘代磷脂、不同种类氘代鞘脂中的一种或多种。所述不同种类氘代磷脂和不同种类氘代鞘脂是指其种类与食品样品中的磷脂和鞘脂的种类不同。例如，所述混合内标可以包括以下中的一种或多种：甘油酯、磷脂酰胆碱、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酰乙醇胺、磷脂酰甘油、磷脂酸、鞘磷脂、神经酰胺等。 所述混合内标能够消除不同实验人员、不同批次样本间由于操作差异和仪器状态差异导致的数据难以统一分析的缺点，具有提高数据可比性和准确性的优点。
所述混合内标的量的确定原则为：混合内标加入后的各脂质的质量浓度为原样品中同种类脂质的质量浓度的60-140％，优选为80％-120％，更优选地，混合内标加入后的各脂质的质量浓度与原样品中同种类脂质的质量浓度相当；即将食品样品检测结果通过外标曲线确定各脂质含量，混合内标中各脂质的添加量为通过外标曲线确定的各脂质的含量的60-140％；优选为80％-120％；更优选为100％。
本发明采用二氯甲烷代替脂质提取、分离、纯化过程中所用的氯仿，提高方法的安全性，二氯甲烷是甲烷氯化物中毒性最小的，其毒性仅为四氯化碳毒性的0.11％。同时其具有与脂质互溶可用于脂质提取、分离、纯化的优点。
在本发明的另一个实施方案中，本发明所述食品脂质提取方法包括以下步骤：将二氯甲烷与甲醇的混合物作为脂质提取溶剂进行第一次离心，得上层相和下层有机相。在本发明的一个实施方案中，所述二氯甲烷、甲醇和超纯水的体积比为1-3.5:1-3:0.2-0.55，优选为1-3:1-3:0.2-0.55，更优选为：9:10:2。通过上述比例的二氯甲烷、甲醇和超纯水进行离心分离后，能够将神经节苷脂分离到上层相中，具有将脂质充分提取并实现脂肪酸/甘油酯/磷脂与神经节苷脂分离的优点。在本发明的一个实施方案中，二氯甲烷的用量为0.5-2mL/100μL液态食品样品或1-3mL/0.05g粉末固体食品样品；优选为0.6-1.2mL/100μL液态食品样品或1.5-2.5mL/0.05g粉末固体食品样品。在该用量下，能够将脂质充分提取，且具有分离不同脂质的优点。
在本发明的一个实施方案中，所述超纯水与二氯甲烷分两次加入，所述甲醇一次添加完毕。在本发明的一个实施方案中，在将食品样品与混合内标混合后，向其中加入超纯水、二氯甲烷和甲醇，摇匀震荡后，再次向 其中加入剩余的超纯水和二氯甲烷。在本发明的一个实施方案中，所述超纯水和二氯甲烷的两次加入量相同。在本发明的一个实施方案中，所述超纯水和二氯甲烷的两次加入量不同。在本发明的一个实施方案中，所述超纯水的两次加入量的比例为0.15-0.4:0.1-0.3；更优选为0.2:0.25。在本发明的一个实施方案中，所述二氯甲烷的两次加入量的比例为0.8-1.0:0.85-1.1；更优选为0.9:1.0。在本发明的一个实施方案中，在加入超纯水、二氯甲烷和甲醇后，摇匀震荡，并进行超声处理，然后再加入剩余的超纯水和二氯甲烷。在本发明的一个实施方案中，所述超声处理在如下条件中进行：20-50KHz，600-900W，温度为20-30℃，超声1-10分钟；优选为40KHz，800W，25℃，超声5分钟。
在本发明的一个实施方案中，所述食品脂质提取方法还包括以下步骤：取第一次离心所得上层相，向其中加入二氯甲烷，以进行第二次离心，得到上层相以及下层有机相。在本发明的一个实施方案中，所述二氯甲烷的用量为0.2-1.8mL/100μL液态食品样品或1-3mL/0.05g粉末固体食品样品。在本发明的一个优选实施方案中，所述二氯甲烷的用量为0.6-1.2mL/100μL液态食品样品或1.5-2.5mL/0.05g粉末固体食品样品。通过上述第二次分离，能够将样品中的脂肪酸/甘油酯/磷脂充分提取到有机相中，具有将不同脂质分离的优点。
在本发明的一个实施方案中，所述食品脂质提取方法还包括以下步骤：取第一次离心所得下层有机相，向其中加入超纯水、甲醇和二氯甲烷，以进行第三次离心，得到上层相以及下层有机相。在本发明的一个实施方案中，所述超纯水、甲醇和二氯甲烷的体积比为0.3-1:1-3.5:0.5-3，优选为：0.3-0.6:1-2:0.5-1。本发明的一个实施方案中，所述二氯甲烷的用量为0.3-1mL/100μL液态食品样品或0.3-1mL/0.05g粉末固体食品样品。通过上述第三次分离，能够将有机相中残留的神经节苷脂充分提取到水相中，具有将神经节苷脂充分提取的优点。
在本发明的另一个实施方案中，所述食品脂质提取方法还包括以下步 骤：将第二次离心得到的上层相与第三次离心得到的上层相合并，氮气吹干，即得神经节苷脂。本发明通过对脂质提取溶剂的限定，将所述神经节苷脂分离到上层相中，与脂肪酸/甘油酯/磷脂分离，能够得到仅含神经节苷脂的提取液，具有高效分离的优点。
在本发明的一个实施方案中，所述食品脂质提取方法还包括纯化所述神经节苷脂的步骤。在本发明的一个实施方案中，所述纯化所述神经节苷脂的步骤具体为：将所得神经节苷脂溶解于60-90％的甲醇溶液中，然后离心，取上层相，氮气吹干，即得所述神经节苷脂。在本发明的另一个实施方案中，所述甲醇溶液的用量为100-350μL/100μL液态食品样品或100-350μL/0.05g粉末固体食品样品。通过上述纯化步骤，本发明得到的神经节苷脂中不含有提取过程中产生的蛋白等沉淀杂志，具有避免杂志进入到液质体系的优点。
在本发明的另一个实施方案中，所述食品脂质提取方法还包括以下步骤：将第二次离心得到的下层有机相与第三次离心得到的下层有机相合并，氮气吹干，即得磷脂、脂肪酸和甘油酯。本发明得到的磷脂、脂肪酸和甘油酯不含蛋白沉淀等难溶性杂志，具有保护液质体系的优点。
在本发明的一个实施方案中，所述食品脂质提取方法进一步包括纯化所述磷脂、脂肪酸和甘油酯的步骤。在本发明的一个实施方案中，所述纯化所述磷脂、脂肪酸和甘油酯的步骤具体为：将所得磷脂、脂肪酸和甘油酯溶解于乙酸铵的二氯甲烷和甲醇溶液中，然后离心，取下层有机相，氮气吹干，即得所述磷脂、脂肪酸和甘油酯。在本发明的一个实施方案中，所述乙酸铵的二氯甲烷和甲醇溶液中的二氯甲烷和甲醇的比例为0.6-1.5:0.6-1.2；优选为1:1。在本发明的一个实施方案中，所述乙酸铵的浓度为5-15mmol/L，优选为8-12mmol/L，更优选为10mmol/L。在本发明的一个实施方案中，所述乙酸铵的二氯甲烷和甲醇溶液的用量为0.2-2.5mL/100μL液态食品样品或0.5-2.5mL/0.05g粉末固体食品样品。
传统的Folch法将样品稀释后，主要采用氯仿:甲醇＝2:1的溶剂提取 样品脂质，离心将下层有机相分离后，上层相中再加入氯仿进行二次提取。本发明中，将液体样品稀释后，脂质提取溶剂为特定体积比的二氯甲烷:甲醇，目的在于将其中的神经节苷脂分离到上层相中；得到的下层有机相和上层相再分别分离：有机相采用特定体积比的二氯甲烷:甲醇:水再次提取其中的神经节苷脂，水相采用二氯甲烷再次提取其中的脂肪酸、甘油酯和磷脂。
在本发明的另一个实施方案中，所述食品脂质提取方法还包括如下步骤：将所得磷脂、脂肪酸和甘油酯溶解于体积比为0.6-1.5:0.6-1.2(优选为2:1)的二氯甲烷和甲醇溶液中，得复溶脂质溶液，然后将所述复溶脂质溶液采用硅胶柱进行分离，分别得到所述甘油酯/脂肪酸以及所述磷脂。在本发明的一个实施方案中，所述二氯甲烷和甲醇溶液的用量为0.2-2.5mL/100μL液态食品样品或0.5-2.5mL/0.05g粉末固体食品样品。
在本发明的另一个实施方案中，所述硅胶柱分离具体包括如下步骤：
活化硅胶柱：用2-5mL正己烷活化硅胶柱(6mL体积，1g吸附剂)Silica gel bonded cartridges(Dikma ProElut Si,6-mL volume,1g sorbents)，弃去洗脱液，然后将所述复溶脂质溶液倒入柱子中；
洗脱非极性脂：首先向柱子中加入1-5mL的正己烷/二乙醚(8:2，V/V)溶液，收集洗脱液，然后加入2-5mL正己烷/二乙醚(1:1，V/V)溶液，收集洗脱液；氮吹干后，即得所述甘油酯/脂肪酸；
洗脱磷脂：向柱子中加入2-5mL的甲醇，然后加入1-3.5mL甲醇和1-3.5mL二氯甲烷/甲醇/水(3:5:2，V/V/V)，收集洗脱液，氮气吹干后，即得所述磷脂。
本发明所述方法同时提取食品中的脂肪酸、甘油酯、磷脂和神经节苷脂，能够同时实现脂质的高效提取与分离，具有样本用量少、提取效率高且实现不同脂质分离的优点。
在另一个实施方案中，本发明提供了一种食品脂质提取方法，其可分别提取脂肪酸/甘油酯/磷脂与神经节苷脂，所述方法包括以下步骤：
1)取100-350μL液态或0.05-0.15g粉末固体食品样品加入1#离心管，向其中加入与所述食品中各脂质含量相当混合内标、100-300μL超纯水、1-3mL甲醇和500-1500μL二氯甲烷，摇匀震荡后进行超声处理，然后再次向其中加入100-250μL超纯水和500-1500μL二氯甲烷，并摇匀震荡；将所得液体离心，分别得上层相和下层有机相；
2)取1#离心管中的上层相加入至2#离心管，向其中加入1-3ml二氯甲烷，并进行离心，分别得上层相和下层有机相；
3)取1#离心管中的下层有机相加入至3#离心管，并向其中加入0.5-3mL超纯水、1-3.5mL甲醇和0.3-1.0mL二氯甲烷，并摇匀震荡；将所得液体离心，得上层相和下层有机相；
4)合并2#离心管和3#离心管中的上层相，氮气吹干后，即得所述神经节苷脂；
5)合并2#离心管和3#离心管中的下层有机相，氮气吹干后，即得所述磷脂、脂肪酸和甘油酯。
在另一个实施方案中，本发明提供了一种食品脂质提取方法，其可分别提取脂肪酸/甘油酯、磷脂、神经节苷脂，所述方法包括以下步骤：
1).取100-350μL液态或0.05-0.15g粉末固体食品样品加入1#离心管，向其中加入与所述食品中各脂质含量相当混合内标、100-350μL超纯水、1-3.5mL甲醇和500-1500μL二氯甲烷，摇匀震荡后进行超声处理，然后再次向其中加入100-350μL超纯水和500-1500μL二氯甲烷，并摇匀震荡；将所得液体离心，分别得上层相和下层有机相；
2).取1#离心管中的上层相加入至2#离心管，向其中加入1-3ml二氯甲烷，并进行离心，分别得上层相和下层有机相；
3).取1#离心管中的下层有机相加入至3#离心管，并向其中加入0.5-3mL超纯水、1.5-3mL甲醇和0.3-1.0mL二氯甲烷，并摇匀震荡；将所得液体离心，得上层相和下层有机相；
4)合并2#离心管和3#离心管中的上层相，氮气吹干后，即得所述神 经节苷脂；
5)合并2#离心管和3#离心管中的下层有机相，氮气吹干后，即得所述磷脂和甘油酯、脂肪酸。
6).将步骤5)所得磷脂、脂肪酸和甘油酯溶解到0.5-2.5ml二氯甲烷:甲醇(v/v＝2:1)中，得复溶脂质溶液，然后采用硅胶柱进行分离，分别得到所述甘油酯/脂肪酸和所述磷脂。
本发明同时提供了一种检测食品脂质的方法；其可具体检测磷脂/甘油酯/脂肪酸以及神经节苷脂。
在本发明的一个实施方案中，所述检测食品脂质的方法包括如下步骤：
1)磷脂/甘油酯/脂肪酸的检测：取上述食品脂质提取方法中提取得到的脂质或甘油酯/脂肪酸、磷脂，将其稀释至脂质可定量检测的上限和下限之间，采用LC-Q-TOF-MS(AB Sciex
Figure PCTCN2019095969-appb-000003
6600)在如下条件中进行检测：色谱柱为：Kinetex C18
Figure PCTCN2019095969-appb-000004
柱(150mm×4.6mm,2.6μm；Phenomenex,Torrance,CA,USA)或者Kinetex C18
Figure PCTCN2019095969-appb-000005
柱(50mm×3mm,2.6μm；Phenomenex,Torrance,CA,USA)；柱温为30-50℃；流动相A为5nmol/L的乙酸铵溶液(甲醇\乙腈\水，1\1\1，V\V\V)，流动相B为5nmol/L的乙酸铵溶液(异丙醇)，流速0.3-0.8mL/min，进样量1-3μL。
在本发明的一个实施方案中，在所述检测对象为上述食品脂质提取方法中提取得到的脂质时，所述检测食品脂质的方法中，所述磷脂/甘油酯/脂肪酸的检测按如下表进行洗脱梯度：
Figure PCTCN2019095969-appb-000006
在本发明的一个实施方案中，在所述检测对象为上述食品脂质提取方法中提取得到的磷脂和/或甘油酯/脂肪酸时，所述检测食品脂质的方法按如下表进行洗脱梯度：
Figure PCTCN2019095969-appb-000007
当样品中磷脂含量与甘油酯含量较高时，可在液相色谱柱后采用切换阀，在一定的保留时间后，磷脂出峰完毕时，将液相色谱柱中洗脱的甘油三酯排到废液缸中，以免进入质谱因离子浓度过高而污染质谱。
质谱在正离子模式和负离子模式下分别检测，正负离子模式下，母离子检测参数：累积时间(accumulation time)0.15secs，扫描范围(tof masses)100-1300Da，洗脱时间(duration)15min，加热气(GS1)50-65psi，辅助加热气(GS2)50-65psi，气帘气(CUR)30-45psi，离子源温度(TEM)500-650℃,离子喷雾电压(ISVF)4500-5500V(若负离子模式，仅将此参数改为-4500V)，去簇电压(DP)80V，碰撞能量(CE)10V。质谱二级碎片检测参数：累积时间(accumulation time)0.05secs，扫描范围(tof masses)50-1300Da，加热气(GS1)50-65psi，辅助加热气(GS2)50-65psi，气帘气(CUR)30-45psi，离子源温度(TEM)500-650℃,离子喷雾电压(ISVF)4500-5500V(若负离子模式，仅将此参数改为-4500V)，碰撞能量(CE)30-50V，碰撞能量叠加(CES)10-25V。
在本发明的一个实施方案中，所述检测食品脂质的方法包括如下步骤：
2)神经节苷脂的检测：取上述食品脂质提取方法中提取得到的神经节苷脂，将其稀释至脂质可定量检测的上限和下限之间，采用 LC-Q-TOF-MS(AB Sciex
Figure PCTCN2019095969-appb-000008
6600)在如下条件中进行检测：色谱柱：BEH C18(100×2.1mm，1.7um；Waters)，柱温为30-50℃；流动相A为90ml水+10ml甲醇+0.1ml 1mol/L的乙酸铵，流动相B为100ml甲醇+0.1ml 1mol/L的乙酸铵，流速0.3mL/min，进样量为10μL。
在本发明的一个实施方案中，所述检测食品脂质的方法中，所述神经节苷脂的检测按如下表进行洗脱梯度：
Figure PCTCN2019095969-appb-000009
质谱模式为负离子模式，母离子检测参数：累积时间(accumulation time)0.25secs，扫描范围(tof masses)100-2000Da，洗脱时间(duration)14min，加热气(GS1)35-50psi，辅助加热气(GS2)30-45psi，气帘气(CUR)10-25psi，离子源温度(TEM)300-450℃，离子喷雾电压(ISVF)-3500-4500V，去簇电压(DP)-30-50V，碰撞能量(CE)-35-50V。质谱二级碎片检测参数：累积时间(accumulation time)0.05secs，扫描范围(tof masses)50-2000Da，加热气(GS1)35-50psi，辅助加热气(GS2)30-45psi，气帘气(CUR)10-25psi，离子源温度(TEM)300-450℃,离子喷雾电压(ISVF)-3500-4500V，去簇电压(DP)-35-50V，碰撞能量(CE)-35-45V，-碰撞能量叠加(CES)15-30V，离子释放延迟(IRD)30，离子释放宽度(IRW)15。
本发明同时提供了脂质定性分析方法，其中所述定性分析均采用Peakview2.2软件。其中，所述脂质定性分析方法包括如下：
1)脂肪酸定性分析：在负离子模式下，将加和离子设置为-H后，直接将离子信息与脂肪酸数据库比较确定。
2)甘油酯定性分析：甘油酯在正离子模式下为+NH 4 +的正离子，根据此软件中自带的甘油酯数据库，首先根据母离子的精确m/z值定性分析，然后根据二级碎片(甘油二酯)信息确定脂肪酸组成。若碎片定性到的脂肪酸可组合出母离子甘油酯中碳原子和双键的数目，则定性此甘油酯；
3)磷脂定性分析：不同的磷脂种类定性方法不同，其中PA、PG、PI、PS、PE均可在负离子模式下根据Peakview自带的数据库导入相应的数据后，将加和离子设置为-H后，PC的加和离子为CH 3COO -，首先根据母离子定性分析，然后根据碎片确定脂肪酸组成是否与相应母离子中碳原子数和双键数一致，若一致则可定性此磷脂。SM和Cer在正离子模式下分析，SM加和离子为H +时由184二级特征碎片定性分析，Cer由264二级特征碎片做定性分析；
4)神经节苷脂定性分析：神经节苷脂在负离子模式下根据Peakview自带的神经节苷脂数据库导入相应的数据后，首先根据精确分子量、同位素和分子式进行检索，检索到的神经节苷脂再根据二级碎片中是否有唾液酸(质核比为290.1)的碎片确定是否含有某特定的神经节苷脂。
本发明同时提供了脂质定量分析方法，其中所述定量分析均采用MultieQuant 3.0.2软件对不同的脂质进行定量分析。其中，所述脂质定量分析方法包括如下：
1)甘油酯定量分析：因脂质前处理过程需人工操作，随机误差不稳定，因此不同甘油酯定量主要采用内标法，内标物含9种甘油酯的氘代内标混合物，添加量据所测乳品中对应甘油酯的含量确定，以内标含量与乳品中对应甘油酯含量相似为原则，其他甘油酯选择保留时间相近的氘代内标计算含量。同时，采用外标法，以不同代表性外标标品与内标的面积比值为纵坐标，不同外标浓度为横坐标建立标准曲线，用于定量计算样品中不同甘油酯的含量；
2)磷脂定量分析：不同种类母乳磷脂定量分析同时采用内标法和外标法。内标法采用磷脂酰胆碱(Phosphatidylchaline,PC)31：2(17:1；14:1)、 磷脂酰丝氨酸(Phosphatidylserine,PS)31:2(17:0-14:1)、磷脂酰肌醇(Phosphatidylinositol,PI)31:2(17:0-14:1)、磷脂酰乙醇胺(Phosphatidylehanolmine,PE)31:2(17:0-14:1)、磷脂酰甘油(phosphatidylglycerol，PG)31:1(17:0-14:1)、磷脂酸(Phosphatidic acid，PA)、鞘磷脂(sphingomyelin，SM)d18:1/17:0、神经酰胺(Ceramide(d18:2(4E,8Z)/24:0))作为内标。同时每种极性脂选择一种作为外标，以外标标品与相应内标的面积比值为纵坐标，以外标浓度为纵坐标建立标准曲线，用于计算样品中不同磷脂的含量；
3)神经节苷脂定量分析：神经节苷脂主要定量其中含量较高的单唾液酸神经节苷脂(Monosialoganglioside，GM3)和双唾液酸神经节苷脂(Disialoganglioside，GD3)，采用外标法定量。GM3和GD3标品中都同时含有多种不同烷基烃和糖组成的单体，因此将其中含量较高峰面积总和作为定量计算的依据。样品中的神经节苷脂定量，同样将不同的GM3和GD3总和计算相应的含量。神经节苷脂GM3和GD3的标准曲线分别如图1和图2所示。
通过同一检测平台LC-Q-TOF-MS(AB Sciex
Figure PCTCN2019095969-appb-000010
6600)，采用合适色谱柱、流动相及洗脱梯度，将非极性的甘油酯、极性磷脂和神经节苷脂洗脱后进入质谱检测，是一针进样同时检测甘油酯、磷脂和神经节苷脂的关键。本发明建立两种方法，一种方法可同时检测出甘油酯、脂肪酸、磷脂和神经节苷脂，用于提取到的同时含有不同种类脂质样品的检测，也可仅检测甘油酯和/或磷脂；另一种方法仅检测神经节苷脂，用于提取到的神经节苷脂样品的检测。
本发明的有益效果
本发明的优点主要体现在以下几个方面：
1、可通过一次取样及处理，将其中的甘油酯、脂肪酸、磷脂和神经节苷脂提取出
文献现有方法神经节苷脂单独提取，且仅有少数文献将磷脂和甘油 酯、脂肪酸共同提取。本发明通过一次取样处理可根据检测分析的需要，还可分别得到神经节苷脂提取物、磷脂/脂肪酸/甘油酯提取物两种；还可根据样品中磷脂、脂肪酸和甘油酯的浓度，为提高磷脂、脂肪酸和甘油酯定性分析和定量分析的准确性，并减少对仪器的污染，可将甘油酯、脂肪酸和磷脂分离，以稀释或浓缩不同的倍数用于进一步的检测。
2、本发明采用二氯甲烷代替氯仿提取脂质，提高实验安全性。
二氯甲烷是甲烷氯化物中毒性最小的，其毒性仅为四氯化碳毒性的0.11％。
文献报道方法中，按照传统的folch法，一个样品需用到60-80ml氯仿，且在提取环节需摇匀、离心，固相萃取时需用氯仿洗脱，而上述环节难以做到完全封闭操作，因此在大批量样品检测过程中，不仅对操作人员和所在实验室人员造成健康影响，废弃的氯仿在存放、运输和处理过程中会造成二次毒害。本发明采用二氯甲烷代替传统方法中的氯仿。
3、建立LC-Q-TOF-MS同时检测甘油酯、磷脂和神经节苷脂的方法
文献报道中，多数是不同脂类分别检测，本发明建立一种LC-Q-TOF-MS同时检测甘油酯、磷脂、神经酰胺、鞘脂的方法，并单独建立一种适合于神经节苷脂提取物的检测方法。
4、此方法适用于不同食品，包括液体状和固体状，尤其是母乳、乳制品及相应配料。
文献报道中的方法多是对不同原料中磷脂或甘油酯或神经节苷脂的比较，同时适合于多种原料的前处理方法还未见报道。
附图说明
图1为：一种神经节苷脂GM3的标准曲线，横坐标为浓度，纵坐标为离子强度。
图2为：另一种神经节苷脂GD3的标准曲线，横坐标为浓度，纵坐标为离子强度。
具体实施方式
在以下的实施例中提供了本发明的示例性的实施方案。以下的实施例仅通过示例的方式给出，并用于帮助普通技术人员使用本发明。所述实施例并不能以任何方式来限制本发明的范围。
试验例1：检测方法的精密度
白天、晚上分别间隔1h，重复进样6次，确定方法的精密度，不同浓度的相对标准偏差(relative standard deviation，RSD)如下表：
表2方法精密度
Figure PCTCN2019095969-appb-000011
准确度
甘油酯/磷脂与神经节苷脂分别提取时，出神经节苷脂外回收率较为合理(表3)，可在不加入内标时，用外标法直接定量。其他两种提取方法需加入内标，用于定量分析样品中的各种脂质含量。神经节苷脂可计算相对含量。
表3回收率
Figure PCTCN2019095969-appb-000012
检出限和定量限
表4检出限和定量限
Figure PCTCN2019095969-appb-000013
实施例1：母乳甘油酯、脂肪酸、磷脂和神经节苷脂分别提取
1样品前处理
1#管：200μL母乳+200μL超纯水+1.8mL甲醇+800μL二氯甲烷(色谱纯)→手动摇匀震荡10s，超声5min→加入200μL超纯水+800μL二氯甲烷(色谱纯)→手动摇匀震荡10s→离心，6000r/min，15min→取出下层有机相到2#管→
1)1#管上层+1.6mL二氯甲烷→离心，6000r/min，15min→取出下层有机相到3#管
2)2#管下层有机相+1mL超纯水+2.2mL甲醇+0.6mL二氯甲烷，手动摇匀→离心，3000g/min，10min→取出下层有机相到3#管混合→氮气吹干→溶解到1ml二氯甲烷：甲醇(v/v＝2:1)中，用于固相萃取分离甘油酯、脂肪酸和磷脂：
活化硅胶柱：用3mL正己烷活化硅胶柱(6mL体积，1g吸附剂 )Silica  gel bonded cartridges(Dikma ProElut Si,6-mL volume,1g sorbents)，弃去洗 脱液，然后将复溶后的脂质溶液倒入柱子中；
洗脱非极性脂：首先向柱子中加入3mL的正己烷/二乙醚(8:2，V/V)溶液，收集洗脱液，然后加入3mL正己烷/二乙醚(1:1，V/V)溶液，收集洗脱液；氮吹干后，2mL复溶液复溶，取出100μL，加入900μL复溶液，6000r/min，5min离心后，取上清上机检测甘油酯和游离脂肪酸；
洗脱磷脂：向柱子中加入4mL的甲醇，然后加入2mL甲醇和2mL二氯甲烷/甲醇/水(3:5:2，V/V/V)，收集洗脱液，氮气吹干后，加入200μL复溶液，6000r/min，5min离心后，取上清上机检测磷脂。
3)取出1#管上层相与2#管上层混合，氮气吹干后，用80％的甲醇200μL复溶，6000r/min，5min离心后，取上清上机检测。
2液相洗脱：采用Kinetex C18
Figure PCTCN2019095969-appb-000014
柱(150mm×4.6mm,2.6μm；Phenomenex,Torrance,CA,USA)色谱柱检测磷脂，柱温35℃，液相梯度洗脱
表5实施例1的洗脱梯度
Figure PCTCN2019095969-appb-000015
采用Kinetex C18
Figure PCTCN2019095969-appb-000016
柱(50mm×3.0mm,2.6μm；Phenomenex,Torrance,CA,USA)色谱柱检测甘油三酯，柱温35℃，流速0.6ml/min，液相梯度洗脱：0min，50％A；2min，50％A；3min,2％A；8min，2％A；8.1min,50％A；10min，50％A。
3质谱检测
质谱在正离子模式和负离子模式下分别检测，正负离子模式下，母离子检测参数：累积时间(accumulation time)0.15secs，扫描范围(tof masses)100-1300Da，洗脱时间(duration)15min，加热气(GS1)60psi，辅助加 热气(GS2)60psi，气帘气(CUR)35psi，离子源温度(TEM)600℃,离子喷雾电压(ISVF)5500V(若负离子模式，仅将此参数改为-4500V)，去簇电压(DP)80V，碰撞能量(CE)10V。质谱二级碎片检测参数：累积时间(accumulation time)0.05secs，扫描范围(tof masses)50-1300Da，加热气(GS1)60psi，辅助加热气(GS2)60psi，气帘气(CUR)35psi，离子源温度(TEM)600℃,离子喷雾电压(ISVF)5500V(若负离子模式，仅将此参数改为-4500V)，碰撞能量(CE)40V，碰撞能量叠加(CES)20V。
4质谱结果分析
定性分析各种脂质及其脂肪酸组成，并相对定量分析各种脂质的含量。
1)检测到游离脂肪酸21种，含量较高的几种依次为:18:2(31.39％)，18:1(31.22％)，14:0(12.12％)，16:0(9.83％)，18:0(7.84％)；
2)甘油二酯46种，甘油三酯97种，含量较高的几种依次为:TAG52:3(9.57％，18:2-18:1-16:0)，TAG52:2(7.87％，18:2-18:0-16:0)，TAG54:5(6.46％,18:2-18:2-18:1)，TAG52:4(6.25％,18:2-18:2-16:0)，TAG54:4(6.18％，18:1-18:1-18:2)
3)甘油磷脂包括：磷脂酰胆碱12种，溶血磷脂酰胆碱2种，磷脂酰乙醇胺24种，溶血磷脂酰乙醇胺8种，磷脂酰肌醇7种，溶血磷脂酰肌醇1种，磷脂酸4种，磷脂酰甘油1种，磷脂酰丝氨酸7种。含量较高的几种依次为：PE36:2(35.45％，18:0-18:2)，PS36:2(10.63％，18:0-18:2)，PE36:1(9.25％，18:0-18:1)，PC36:2(8.37％，18:0-18:2)，PE36:3(4.20％，18:1-18:2)。
4)鞘脂类鞘磷脂19种，神经酰胺11种，含量较高的几种依次为：SM 40:1；2(31.74％)，SM 42:1；2(30.72％)，SM 42:2；2(13.47％)，SM 38:1；2(8.56％)，SM 36:1；2(5.14％)。
5)糖脂25种，含量较高的几种依次为：GM3 36:1；2(20.25％)，GM3  40:1；2(16.79％)，GM3 38:1；2(16.76％)，GM3 42:2；2(16.20％)，GM3 34:1；2(10.17％)。
实施例2同时提取母乳脂质
1样品前处理
80uL母乳+1200uL超纯水+2.5mL甲醇+1200uL二氯甲烷(色谱纯)→手动摇匀震荡10S，静置30min→加入1.5mL超纯水+1mL二氯甲烷(色谱纯)→手动摇匀震荡10S→离心，6000r/min,15min→取出下层有机相→上层+1.6mL二氯甲烷→离心，6000r/min,15min→取出下层有机相与之前的有机相混合→氮吹干→250uL 10mmol/L乙酸铵(v/v，二氯甲烷：甲醇＝1:1)溶液溶解→离心，6000r/min，5min。
2采用Kinetex C18
Figure PCTCN2019095969-appb-000017
柱(150mm×4.6mm,2.6μm；Phenomenex,Torrance,CA,USA)色谱柱，柱温40℃，液相梯度洗脱
表6实施例2的液相洗脱梯度
Figure PCTCN2019095969-appb-000018
3质谱检测
质谱在正离子模式和负离子模式下分别检测，正负离子模式下，母离子检测参数：累积时间(accumulation time)0.15secs，扫描范围(tof masses)100-1300Da，洗脱时间(duration)15min，加热气(GS1)55psi，辅助加热气(GS2)50psi，气帘气(CUR)30psi，离子源温度(TEM)550℃,离子喷雾电压(ISVF)5000V，去簇电压(DP)70V，碰撞能量(CE)10V。质谱二级碎片检测参数：累积时间(accumulation time)0.05secs，扫描范围(tof masses)50-1300Da，加热气(GS1)55psi，辅助加热气(GS2)50psi，气帘气(CUR)30psi，离子源温度(TEM)550℃,离子喷雾电压 (ISVF)5000V，碰撞能量(CE)35V，碰撞能量叠加(CES)15V。
4质谱结果分析(需有相对定量的结果)
最终检测游离脂肪酸7种，甘油酯76种(单甘脂2种、甘油二酯15种、甘油三酯59种)、磷脂42种(PC12种、PA3种、PE10种、PG4种、PS1种、SM8种、Cer4种)、糖脂2种。
实施例3母乳甘油酯/脂肪酸/磷脂和神经节苷脂分别提取，两次检测
1样品前处理
100μL母乳到1#管+150μL超纯水+1.5mL甲醇+800μL二氯甲烷(色谱纯)→手动摇匀震荡10S，超声5min→加入200μL超纯水+800μL二氯甲烷(色谱纯)→手动摇匀震荡10S→离心，6000r/min，15min→取出上层相到2#管：
步骤1：2#管+1.6mL二氯甲烷→离心，6000r/min，15min→取出上层相到3#管，等着与步骤2中取出的上层相混合
步骤2：1#管剩下有机相+2mL甲醇+0.8mL二氯甲烷+1mL超纯水→离心，6000r/min，15min→取出上层相与步骤1中3#管的上层混合后，上层相直接过滤上样检测神经节苷脂
步骤3：从步骤2中的1#管中取出有机相与2#管中的有机相混合→氮吹干→一定量10mmol/L乙酸铵(V/V,二氯甲烷：甲醇＝1:1)溶液溶解→离心，6000r/min，5min，上清用于检测磷脂、脂肪酸和甘油酯
2采用Kinetex C18
Figure PCTCN2019095969-appb-000019
柱(150mm×4.6mm,2.6μm；Phenomenex,Torrance,CA,USA)色谱柱，柱温40℃，液相梯度洗脱
表7实施例3的洗脱梯度
Figure PCTCN2019095969-appb-000020
3质谱检测
质谱在正离子模式和负离子模式下分别检测，正负离子模式下，母离子检测参数：累积时间(accumulation time)0.15secs，扫描范围(tof masses)100-1300Da，洗脱时间(duration)15min，加热气(GS1)65psi，辅助加热气(GS2)65psi，气帘气(CUR)40psi，离子源温度(TEM)650℃,离子喷雾电压(ISVF)5500V，去簇电压(DP)70V，碰撞能量(CE)15V。质谱二级碎片检测参数：累积时间(accumulation time)0.05secs，扫描范围(tof masses)50-1300Da，加热气(GS1)65psi，辅助加热气(GS2)65psi，气帘气(CUR)35psi，离子源温度(TEM)600℃,离子喷雾电压(ISVF)5500V(若负离子模式，仅将此参数改为-4500V)，碰撞能量(CE)40V，碰撞能量叠加(CES)20V。
4质谱结果分析
分析到游离脂肪酸24种，甘油酯144种(甘油二酯44种，甘油三酯100种)，磷脂57种(PC10种、PE27种、PA3种、PG2种、PI 11种、PS4种)、鞘脂22种，糖脂18种。
实施例4牛乳甘油酯/脂肪酸/磷脂和神经节苷脂分别提取
1样品前处理
100μL牛乳到1#管+150μL超纯水+1.5mL甲醇+800μL二氯甲烷(色谱纯)→手动摇匀震荡10S，超声5min→加入200μL超纯水+800μL二氯甲烷(色谱纯)→手动摇匀震荡10S→离心，6000r/min，15min→取出上层相到2#管：
步骤1：2#管+1.6mL二氯甲烷→离心，6000r/min，15min→取出上层相到3#管，等着与步骤2中取出的上层相混合
步骤2：1#管剩下有机相+2mL甲醇+0.8mL二氯甲烷+1mL超纯水→离心，6000r/min，15min→取出上层相与步骤1中3#管的上层混合后，上层相直接过滤上样检测神经节苷脂
步骤3：从步骤2中的1#管中取出有机相与2#管中的有机相混合→ 氮吹干→一定量10mmol/L乙酸铵(V/V,二氯甲烷：甲醇＝1:1)溶液溶解→离心，6000r/min，5min，上清用于检测磷脂、脂肪酸和甘油酯
2采用Kinetex C18
Figure PCTCN2019095969-appb-000021
柱(150mm×4.6mm,2.6μm；Phenomenex,Torrance,CA,USA)色谱柱，柱温35℃，液相梯度洗脱
表8实施例4的洗脱梯度
Figure PCTCN2019095969-appb-000022
3质谱检测
质谱在正离子模式和负离子模式下分别检测，正负离子模式下，母离子检测参数：累积时间(accumulation time)0.15secs，扫描范围(tof masses)100-1300Da，洗脱时间(duration)15min，加热气(GS1)65psi，辅助加热气(GS2)65psi，气帘气(CUR)40psi，离子源温度(TEM)650℃,离子喷雾电压(ISVF)5500V，去簇电压(DP)70V，碰撞能量(CE)15V。质谱二级碎片检测参数：累积时间(accumulation time)0.05secs，扫描范围(tof masses)50-1300Da，加热气(GS1)65psi，辅助加热气(GS2)65psi，气帘气(CUR)35psi，离子源温度(TEM)600℃,离子喷雾电压(ISVF)5500V(若负离子模式，仅将此参数改为-4500V)，碰撞能量(CE)40V，碰撞能量叠加(CES)20V。
4质谱结果分析
1)游离脂肪酸6种，含量最高的几种依次为：18:0(60.91％)，16:0(31.84％)，18:1(4.96％)
2)甘油二酯8种，甘油三酯64种，含量最高的几种依次为：TAG36:0(8.65％，4:0-14:0-18:0)，TAG38:1(8.00％，4:0-16:0-18:1)，TAG38:0(6.60％，4:0-16:0-18:0)，TAG50:1(4.99％，18:1-14:0-18:0)，TAG40:1 (4.95％，6:0-16:0-18:1)
3)磷脂检测到磷脂酰乙醇6种，磷脂酰胆碱1种，含量较高的几种依次为：PE36:1(29.88％，18:1-18:0)，PE36:2(23.24％，18:1-18:1)，PE36:3(18.75％，18:1-18:2)
4)未检测到鞘脂类和糖脂类
实施例5奶粉中甘油酯/脂肪酸/磷脂和神经节苷脂分别提取
1样品前处理
4.3g奶粉溶解到50℃的水中，摇匀充分溶解，取100μL到1#管+150μL超纯水+1.5mL甲醇+800μL二氯甲烷(色谱纯)→手动摇匀震荡10S，超声5min→加入200μL超纯水+800μL二氯甲烷(色谱纯)→手动摇匀震荡10S→离心，6000r/min，15min→取出上层相到2#管：
步骤1：2#管+1.6mL二氯甲烷→离心，6000r/min，15min→取出上层相到3#管，等着与步骤2中取出的上层相混合
步骤2：1#管剩下有机相+2mL甲醇+0.8mL二氯甲烷+1mL超纯水→离心，6000r/min，15min→取出上层相与步骤1中3#管的上层混合后，上层相直接过滤上样检测神经节苷脂
步骤3：从步骤2中的1#管中取出有机相与2#管中的有机相混合→氮吹干→一定量10mmol/L乙酸铵(V/V,二氯甲烷：甲醇＝1:1)溶液溶解→离心，6000r/min，5min，上清用于检测磷脂、脂肪酸和甘油酯
2采用Kinetex C18
Figure PCTCN2019095969-appb-000023
柱(150mm×4.6mm,2.6μm；Phenomenex,Torrance,CA,USA)色谱柱，柱温35℃，液相梯度洗脱
表9实施例5的洗脱梯度
Figure PCTCN2019095969-appb-000024
3质谱检测
质谱在正离子模式和负离子模式下分别检测，正负离子模式下，母离子检测参数：累积时间(accumulation time)0.15secs，扫描范围(tof masses)100-1300Da，洗脱时间(duration)15min，加热气(GS1)65psi，辅助加热气(GS2)65psi，气帘气(CUR)40psi，离子源温度(TEM)650℃,离子喷雾电压(ISVF)5500V，去簇电压(DP)70V，碰撞能量(CE)15V。质谱二级碎片检测参数：累积时间(accumulation time)0.05secs，扫描范围(tof masses)50-1300Da，加热气(GS1)65psi，辅助加热气(GS2)65psi，气帘气(CUR)35psi，离子源温度(TEM)600℃,离子喷雾电压(ISVF)5500V(若负离子模式，仅将此参数改为-4500V)，碰撞能量(CE)40V，碰撞能量叠加(CES)20V。
4质谱结果分析
1)奶粉中检测到的游离脂肪酸6种，其中含量较高的几种依次为：18:0(61.26％)，16:0(33.23％)
2)甘油二酯12种，甘油三酯75种，其中含量较高的几种依次为：TAG52:2(8.97％，18:1-16:0-18:1)，TAG54:5(7.61％，18:2-18:2-18:1)，TAG50:1(7.41％，16:0-16:0-18:1)，TAG54:6(7.02％，18:3-18:1-18:2)，TAG54:3(6.36％，18:2-18:2-18:0)。
3)磷脂胆碱4种，磷脂酰乙醇胺6中，磷脂酰肌醇4种，其中含量较高的几种依次为：PE36:2(36.49％，18:1-18:1)，PE36:3(12.64％，18:1-18:2)，PE34:1(10.44％，16:0-18:1)。
4)鞘磷脂和糖脂未检测到。
比较例1
与实施例1所用的方法相比，本比较例采用传统的Folch法提取，具体如下：
100uL母乳+2.5ml的氯仿/甲醇溶液(v/v＝2:1)+250uL NaCl(0.05M)，摇床20min摇匀，200rpm，5000rpm离心20min，取出底层氯仿，上层加 入1.8ml氯仿，摇床20min摇匀，200rpm，5000rpm离心20min，底层氯仿与上次的混合氮吹后，按照实施例1的方法复溶、固相萃取磷脂、脂肪酸和甘油酯并检测，上层氮吹后用实施例1的方法复溶、离心等处理，检测神经节苷脂。同一份母乳样本所得的结果比较如下，从表中可以看出，Folch法与现有的方法相比，游离脂肪酸、甘油酯、甘油磷脂、鞘脂和神经节苷脂提取到的种类数均较少。
  实施例1方法 本实施例方法
检测到游离脂肪酸 21 9
甘油二酯 18 3
甘油三酯 70 25
磷脂酰胆碱 9 5
磷脂酰乙醇胺 35 12
磷脂酰肌醇 9 2
磷脂酸 9 4
磷脂酰甘油 2 0
磷脂酰丝氨酸 7 3
鞘磷脂 19 14
神经酰胺 7 0
神经节苷脂 14 4
工业实用性
本发明提供一种食品脂质提取方法和脂质检测的方法，所述食品脂质提取方法包括将食品样品与混合内标混合，然后与脂质提取溶剂和超纯水进行多次混合以及多次离心，合并多次离心后所得上层相包含神经节苷脂，合并多次离心后所得下层有机相包含磷脂、脂肪酸和甘油酯。本发明采用少量样本，通过较为安全的脂质提取试剂，经过一次处理，得到不同脂质的提取物后，同时或分别对其进行定性和定量检测。以减少样本用量，提高安全性，节约样品预处理和检测过程中的耗材，降低成本并提高效率，具有较好的经济价值和应用前景。

Claims (9)

  1. 一种食品脂质提取方法，包括将食品样品与混合内标混合，然后与脂质提取溶剂和超纯水进行多次混合以及多次离心，合并多次离心后所得上层相包含神经节苷脂，合并多次离心后所得下层有机相包含磷脂和甘油酯、脂肪酸；其中，所述脂质为神经节苷脂、磷脂、甘油酯和脂肪酸；所述方法所述脂质提取溶剂为二氯甲烷或二氯甲烷、甲醇和超纯水的混合物。
  2. 根据权利要求1所述的方法，其中，所述混合内标为氘代甘油酯、不同种类氘代磷脂、不同种类氘代鞘脂中的一种或多种；
    所述混合内标的量由如下方法确定得到：混合内标加入后的各脂质的质量浓度为原样品中同种类脂质的质量浓度的60-140％。
  3. 根据权利要求1或2所述的方法，包括以下步骤：将二氯甲烷与甲醇的混合物作为脂质提取溶剂，并加入超纯水，以进行第一次离心，得上层相和下层有机相；其中，所述二氯甲烷、甲醇和超纯水的体积比为1-3.5:1-3:0.2-0.55，优选为1-3:1-3:0.3-0.55；
    所述二氯甲烷的用量为0.5-2mL/100μL液态食品样品或1-3mL/0.05g粉末固体食品样品；所述二氯甲烷的用量优选为0.6-1.2mL/100μL液态食品样品或1.5-2.5mL/0.05g粉末固体食品样品。
  4. 根据权利要求3所述的方法，包括以下步骤：取第一次离心所得上层相，向其中加入二氯甲烷，以进行第二次离心，得到上层相以及下层有机相；所述二氯甲烷的用量为0.2-1.8mL/100μL液态食品样品或1-3mL/0.05g粉末固体食品样品；优选地，所述二氯甲烷的用量为0.6-1.2mL/100μL液态食品样品或1.5-2.5mL/0.05g粉末固体食品样品；
    取第一次离心所得下层有机相，向其中加入超纯水、甲醇和二氯甲烷，以进行第三次离心，得到上层相以及下层有机相；所述超纯水、甲醇和二氯甲烷的体积比为0.3-1:1-3.5:0.5-3；优选为0.3-0.6:1-2:0.5-1；所述二氯甲烷的用量为0.3-1mL/100μL液态食品样品或0.3-1mL/0.05g粉末固体食 品样品。
  5. 根据权利要求4所述的方法，包括以下步骤：将第二次离心得到的上层相与第三次离心得到的上层相合并，氮气吹干，即得神经节苷脂；
    优选地，所述方法进一步包括纯化所述神经节苷脂的步骤：将所得神经节苷脂溶解于60-90％的甲醇溶液中，然后离心，取上层相，氮气吹干，即得所述神经节苷脂；优选地，所述甲醇溶液的用量为100-350μL/100μL液态食品样品或100-350μL/0.05g粉末固体食品样品。
  6. 根据权利要求4所述的方法，包括以下步骤：将第二次离心得到的下层有机相与第三次离心得到的下层有机相合并，氮气吹干，即得磷脂和甘油酯、脂肪酸；
    优选地，所述方法进一步包括纯化所述磷脂和甘油酯、脂肪酸的步骤：将所得磷脂和甘油酯、脂肪酸溶解于乙酸铵的二氯甲烷和甲醇溶液中，然后离心，取下层有机相，氮气吹干，即得所述磷脂和甘油酯、脂肪酸；
    优选地，所述乙酸铵的二氯甲烷和甲醇溶液中的二氯甲烷和甲醇的比例为0.6-1.5:0.6-1.2；优选为1:1；所述乙酸铵的浓度为5-15mmol/L，优选为8-12mmol/L；所述乙酸铵的二氯甲烷和甲醇溶液的用量为0.2-2.5mL/100μL液态食品样品或0.5-2.5mL/0.05g粉末固体食品样品。
  7. 根据权利要求6所述的方法，包括如下步骤：将所得磷脂、脂肪酸和甘油酯溶解于体积比为0.6-1.5:0.6-1.2的二氯甲烷和甲醇溶液中，得复溶脂质溶液，然后将所述复溶脂质溶液采用硅胶柱进行分离，分别得到所述甘油酯/脂肪酸和所述磷脂；
    优选地，所述二氯甲烷和甲醇溶液的用量为0.2-2.5mL/100μL液态食品样品或0.5-2.5mL/0.05g粉末固体食品样品。
  8. 一种检测食品脂质的方法，包括如下步骤：
    1)磷脂/甘油酯/脂肪酸检测：取权利要求1-7中任一项方法提取得到的所述甘油酯、脂肪酸和磷脂，将其稀释至脂质可定量检测的上限和下限之间，采用LC-Q-TOF-MS(AB Sciex
    Figure PCTCN2019095969-appb-100001
    6600)在如下条件中进 行检测：色谱柱为：Kinetex C18
    Figure PCTCN2019095969-appb-100002
    柱(150mm×4.6mm,2.6μm；Phenomenex,Torrance,CA,USA)或者Kinetex C18
    Figure PCTCN2019095969-appb-100003
    柱(50mm×3mm,2.6μm；Phenomenex,Torrance,CA,USA)；柱温为30-50℃；流动相A为5nmol/L的乙酸铵溶液(甲醇\乙腈\水，1\1\1，V\V\V)，流动相B为5nmol/L的乙酸铵溶液(异丙醇)，流速0.3-0.8mL/min，进样量1-3μL，进行梯度洗脱。
  9. 根据权利要求8所述的检测食品脂质的方法，还包括如下步骤：
    2)神经节苷脂的检测：取权利要求1-7中任一项方法提取得到的所述神经节苷脂，将其稀释至脂质可定量检测的上限和下限之间，采用LC-Q-TOF-MS(AB Sciex
    Figure PCTCN2019095969-appb-100004
    6600)在如下条件中进行检测：色谱柱：BEH C18(100×2.1mm，1.7um；Waters)，柱温为30-50℃；流动相A为90ml水+10ml甲醇+0.1ml 1mol/L的乙酸铵，流动相B为100ml甲醇+0.1ml 1mol/L的乙酸铵，流速0.3mL/min，进样量为10μL，进行梯度洗脱。
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