Method for recovering acidic lipid

Abstract

To provide an acidic lipid recovery method capable of being simple and reliable.SOLUTION: A method for recovery of acidic lipid includes: a preparation step of preparing a sample comprising the acidic lipid; an application step of applying the prepared sample to a strong anion exchange column; a washing step of applying a washing solution to the strong anion exchange column; and a recovery step of applying an elution solution to the strong anion exchange column to recover the acidic lipid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for recovering acidic lipids. [Background technology]

[0002] Lipids that make up biological membranes consist of simple lipids and complex lipids, the latter of which includes phospholipids and glycolipids. Phospholipids and glycolipids consist of glycerolipids, which have a glycerol backbone, and sphingolipids, which have a sphingoid base. Glycerolipids include major phospholipids (glycerophospholipids), such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. These lipids not only serve as structures that maintain the body, but also play important roles as functional components involved in physiological activity. Therefore, there is a need for the development of analytical methods that can be easily measured in small amounts and are compatible with high throughput. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2008-156571 [Non-patent literature]

[0004] [Non-Patent Document 1] Trinidad Perez-Palacios, Jorge Ruiz, Teresa Antequera. Improvement of a solid phase extraction method for separation of animal muscle phospholipid classes. Food Chem. 2007, 102, 875-879. Summary of the Invention [Problem to be solved by the invention]

[0005] In conventional methods, lipid extracts are typically obtained from biological samples such as plant leaves using solvent extraction methods (e.g., Bligh & Dyer, MTBE, or BUME) and then directly measured using a mass spectrometer (Figure 1). However, the concentrations of lipids contained in biological samples vary significantly depending on the type. This makes it difficult to detect low-concentration lipids. For example, Arabidopsis thaliana contains phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI). However, the concentrations of acidic lipids such as PG and PI are lower than those of PC and PE. Therefore, simultaneous quantification of these four lipids limits the accuracy of the quantification (Figure 2). For these reasons, the sensitivity of mass spectrometers varies significantly between lipid types, and the concentration of each lipid type varies significantly within the living organism, significantly limiting the application of mass spectrometry. Gas chromatography is also used to quantify lipids, but because gas chromatography detects fatty acid methyl esters released from lipids, it is not possible to determine the type of lipid from which the fatty acids originate, and therefore lipid fractionation is essential. Therefore, attempts have been made to fractionate lipid components by type and then quantify and analyze each fractionated type.

[0006] Conventional methods for lipid fractionation include fractionating phospholipids by solid-phase extraction, and completely fractionating lipids by type using two-dimensional thin-layer chromatography (TLC) using a mixed solvent mainly consisting of chloroform and methanol.

[0007] A known method using solid-phase extraction is a column packed with a solid phase containing aminopropyl groups (sometimes referred to as an "anion exchange column" or "aminopropyl column"), as described in Non-Patent Document 1. In this method, a lipid-containing sample is loaded onto a conditioned aminopropyl column, followed by elution with (1) acetonitrile:1-propanol (volume ratio 2:1), (2) methanol, (3) 2-propanol:3N hydrochloric acid / methanol (volume ratio 4:1), and (4) chloroform:methanol:3N hydrochloric acid (volume ratio 200:100:1), sequentially separating the phospholipids phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol (see, e.g., Figure 3). However, because solid-phase extraction using an aminopropyl column relies on the combined effects of hydrophilic, hydrophobic, and electrostatic interactions to fractionate, the amount of solid phase and the composition and amount of solvent used must be strictly controlled (see, e.g., Figure 3). Therefore, slight differences in the procedure can affect the success or failure of fractionation, and it is necessary to verify each time.

[0008] Also known is a method for fractionating phospholipids and gangliosides from others using metal oxide, as described in Patent Document 1. However, as in Non-Patent Document 1, this method is unable to eliminate the concentration differences between lipid components in the body, and low-concentration lipids tend to be difficult to detect during measurement.

[0009] On the other hand, lipid fractionation using TLC can separate individual lipids by type, but it is extremely time-inefficient and is strongly affected by atmospheric humidity, making it difficult to reproduce.

[0010] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a simple and reliable method for recovering acidic lipids. [Means for solving the problem]

[0011] As a result of extensive research, the present inventors have discovered that acidic lipids can be recovered simply and reliably by applying a sample containing acidic lipids to a strong anion exchange column, and have completed the present invention.

[0012] A first aspect of the present invention is a method for recovering acidic lipids, comprising: A preparation step of preparing a sample containing the acidic lipid; an application step of applying the prepared sample to a strong anion exchange column; a washing step in which a wash solution is applied to the strong anion exchange column; a recovery step of applying the elution solution to the strong anion exchange column to recover the acidic lipids; Includes:

[0013] A second aspect of the present invention is a method for analyzing acidic lipids in a sample, comprising the steps of: A sample obtaining step of obtaining an analytical sample containing the acidic lipid recovered from the sample by the method for recovering acidic lipids according to the first aspect; a separation step of separating the analytical sample into components by liquid chromatography; an analysis step of analyzing the separated components using a mass spectrometer; Includes:

[0014] A third aspect of the present invention is a kit for recovering acidic lipids, comprising: a strong anion exchange column; A cleaning solution; an elution solution; Includes: [Effects of the Invention]

[0015] According to the present invention, it is possible to provide a simple and reliable method for recovering acidic lipids. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a schematic diagram showing a conventional lipid analysis procedure. [Figure 2]FIG. 2 is a chromatogram showing the results of analyzing lipid components extracted from Arabidopsis leaves by solvent extraction. [Figure 3] FIG. 3 is a flow chart showing an example of a fractionation method using an aminopropyl column. [Figure 4] FIG. 4 is a schematic diagram showing the procedure for analyzing acidic lipids according to this embodiment. [Figure 5] FIG. 5 is a flow chart showing an example of a fractionation method using a strong anion exchange column according to this embodiment. [Figure 6] FIG. 6 is a graph showing the recovery rate of high peak fractions recovered by the fractionation method using a strong anion exchange column according to this embodiment. [Figure 7] FIG. 7 is a graph showing the recovery rate of low peak fractions recovered by the fractionation method using a strong anion exchange column according to this embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0017] An embodiment of the present invention (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited to this. In this specification, the notation in the format "A to Z" means the upper and lower limits of a range (i.e., A or more and Z or less), and when no unit is specified for A and a unit is specified only for Z, the unit of A and the unit of Z are the same.

[0018] <Method for recovering acidic lipids> A first aspect of this embodiment is a method for recovering acidic lipids, comprising: A preparation step of preparing a sample containing the acidic lipid; an application step of applying the prepared sample to a strong anion exchange column; a washing step in which a wash solution is applied to the strong anion exchange column; a recovery step of applying the elution solution to the strong anion exchange column to recover the acidic lipids; Includes:

[0019] <Preparation process> In this step, a sample containing an acidic lipid is prepared. In this embodiment, "acidic lipid" refers to a lipid that releases a proton in an environment of approximately pH 3 or higher and has a negative charge. Examples of acidic lipids include acidic phospholipids and sulfoquinovosyldiacylglycerol. Examples of acidic phospholipids include phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidic acid (PA). In one aspect of this embodiment, the acidic lipid preferably contains at least one selected from the group consisting of phosphatidylglycerol, phosphatidylinositol, and sulfoquinovosyldiacylglycerol.

[0020] In this embodiment, the sample is not particularly limited as long as it contains the acidic lipid. The sample may be derived from an animal or a plant. Examples of animals include humans and mice. Examples of plants include Arabidopsis thaliana and algae. In one aspect of this embodiment, the sample is preferably derived from a plant.

[0021] The method for obtaining a plant-derived sample is not particularly limited and any known method can be used. For example, an extract containing acidic lipids may be obtained from the leaves, stems, roots, etc. of the plant to be analyzed by a solvent extraction method (e.g., Bligh & Dyer method, MTBE method, BUME method, etc.), and used as the sample.

[0022] <Applicable process> In this step, the prepared sample is applied to a strong anion exchange column. Here, the term "strong anion exchange column" refers to a column having ion exchange groups in the stationary phase, which are positively charged in a pH range of 0 to 14.

[0023] Examples of ion exchange groups that are positively charged in a pH range of 0 to 14 include quaternary ammonium groups. In one aspect of this embodiment, the stationary phase of the strong anion exchange column preferably has quaternary ammonium groups. Examples of the quaternary ammonium groups include trimethylammonium groups and dimethylethanolammonium groups.

[0024] The material of the stationary phase (carrier) packed in the strong anion exchange column may be silica gel or a polymer resin (for example, polystyrene divinylbenzene resin, polymethacrylate resin).

[0025] The particle size of the stationary phase packed in the strong anion exchange column is preferably 10 to 100 μm, more preferably 30 to 60 μm. The particle size of the stationary phase can be measured by a known method. If the strong anion exchange column is commercially available, the particle size of the stationary phase can be determined from the catalog provided by the distributor.

[0026] The strong anion exchange column may be a commercially available column. Examples of commercially available strong anion exchange columns include Oasis MAX 30 mg (1 mL cartridge) (manufactured by Waters) and InertSep MAX FF 60 mg (3 mL cartridge) (manufactured by GL Sciences). In one aspect of this embodiment, the strong anion exchange column may be a plate-type column in which a stationary phase is previously provided on the bottom surface of a microplate.

[0027] In this embodiment, the strong anion exchange column may be conditioned before the sample is applied to it. Examples of the solvent or solution used for conditioning include methanol and the washing solution described below.

[0028] In this step, other conditions (for example, column temperature, flow rate, flow rate, etc.) are appropriately set so that components (acidic lipids) contained in the sample can efficiently bind to the strong anion exchange column.

[0029] <Cleaning process> In this step, a washing solution is applied to the strong anion exchange column. The washing solution preferably contains 2-propanol and a buffer solution of a volatile salt. Examples of the volatile salt include ammonium formate and ammonium acetate.

[0030] The concentration of the 2-propanol is preferably 60% by volume or more and 90% by volume or less, and more preferably 70% by volume or more and 80% by volume or less, based on the total volume of the cleaning solution.

[0031] The concentration of the volatile salt is preferably 2.5 mM or more and 50 mM or less, and more preferably 2.5 mM or more and 20 mM or less, based on the total amount of the washing solution.

[0032] In one aspect of this embodiment, the remainder of the cleaning solution is preferably water.

[0033] In this embodiment, the amount (volume) of the washing solution applied to the strong anion exchange column is preferably 3 to 10 times the volume of the strong anion exchange column, and more preferably 3 to 7 times the volume of the strong anion exchange column.

[0034] In this step, other conditions (for example, column temperature, flow rate, etc.) are appropriately set so that acidic lipids are retained on the strong anion exchange column while other components contained in the sample can be efficiently removed.

[0035] In another aspect of this embodiment, the washing step may further include collecting the washing solution that has passed through the strong anion exchange column. This allows lipids other than acidic lipids (e.g., neutral lipids) contained in the collected washing solution to be recovered. Examples of lipids other than acidic lipids include monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), PC, and PE.

[0036] <Recovery process> In this step, an elution solution is applied to the strong anion exchange column to recover the acidic lipids. The elution solution preferably contains 2-propanol and ammonium chloride, ammonium sulfate, ammonium citrate, or ammonium nitrate. In one aspect of this embodiment, the elution solution more preferably contains 2-propanol and ammonium chloride.

[0037] The concentration of the 2-propanol is preferably 70% by volume or more and 90% by volume or less, and more preferably 80% by volume or more and 90% by volume or less, based on the total volume of the elution solution.

[0038] The concentration of the ammonium chloride, ammonium sulfate, ammonium citrate, or ammonium nitrate is preferably 200 mM or more and 1 M or less, more preferably 200 mM or more and 500 mM or less, based on the entire elution solution.

[0039] In one aspect of this embodiment, the remainder of the cleaning solution is preferably water.

[0040] In this embodiment, the amount (volume) of the elution solution applied to the strong anion exchange column is preferably 3 to 7 times the volume of the strong anion exchange column, and more preferably 3 to 5 times the volume of the strong anion exchange column.

[0041] In this step, other conditions (for example, column temperature, flow rate, etc.) are appropriately set so that acidic lipids are retained on the strong anion exchange column while other components contained in the sample can be efficiently removed.

[0042] Conventionally, lipids have been classified by type using a column packed with a solid phase containing aminopropyl groups (anion exchange column) (e.g., Non-Patent Document 1). However, solid-phase extraction using this anion exchange column involves fractionation using a combination of hydrophilic, hydrophobic, and electrostatic interactions, so the amount of solid phase and the composition and amount of the solvent used must be strictly controlled (e.g., Figure 3). Therefore, even slight differences in the procedure can affect the success or failure of fractionation, and each time verification is required. Furthermore, no method was known for recovering lipids contained in trace amounts in a sample (e.g., acidic lipids in plants) at a quantifiable concentration.

[0043] As a result of intensive research, the inventors discovered that acidic lipids can be easily and reliably recovered by applying a strong anion exchange column to a sample containing acidic lipids, and thus completed the present invention. Although the use of a strong anion exchange column for sample recovery intended for mass spectrometry has traditionally been avoided, actual use has revealed that acidic lipids can be separated from other lipids in a "simple and reliable" manner beyond expectations. The recovery method according to this embodiment is suitable for processing multiple samples.

[0044] <Method for analyzing acidic lipids in a sample> A second aspect of this embodiment is a method for analyzing acidic lipids in a sample, comprising: A sample obtaining step of obtaining an analytical sample containing the acidic lipid recovered from the sample by the method for recovering acidic lipids according to the first aspect; a separation step of separating the analytical sample into components by liquid chromatography; an analysis step of analyzing the separated components using a mass spectrometer; Includes:

[0045] <Sample acquisition process> In this step, an analytical sample containing the acidic lipids recovered from the sample is obtained by the method for recovering acidic lipids according to the first embodiment. Here, the "analytical sample" refers to the sample recovered by the method for recovering acidic lipids, and can also be understood as the sample to be subjected to the separation step described below. The details of the method for recovering acidic lipids according to the first embodiment are as described above.

[0046] In one aspect of this embodiment, the sample acquisition step may include drying the sample recovered by the acidic lipid recovery method, and redissolving the dried sample in a predetermined solvent to obtain an analytical sample. The predetermined solvent is not particularly limited as long as it sufficiently dissolves the dried sample, and examples thereof include the solvents used in the examples described below. The method for drying the sample is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include drying in a vacuum and drying with nitrogen gas.

[0047] <Separation process> In this step, the analytical sample is separated into individual components using a liquid chromatograph. Liquid chromatographs (LCs) utilize the differences in the affinities of the components in the analytical sample for the mobile phase and the stationary phase to separate and elute the components at different retention times. The type of liquid chromatograph is not limited as long as it can separate the components to the desired accuracy so that the components can be separated and detected using a mass spectrometer. Examples of liquid chromatographs that can be used include nano-LC, micro-LC, high-performance liquid chromatographs (HPLC), and ultra-high-performance liquid chromatographs (UHPLC).

[0048] In this embodiment, the mobile phase is not particularly limited, but examples thereof include a mixed solvent of ammonium formate and acetonitrile as mobile phase A, 2-propanol as mobile phase B, and the mobile phases described in the Examples below.

[0049] In this embodiment, the stationary phase is not particularly limited, but examples thereof include silanes to which linear hydrocarbons such as C8 and C18 are bonded, supported on a carrier such as silica gel, and stationary phases described in the examples below.

[0050] <Analysis process> In this step, the separated components are analyzed using a mass spectrometer.

[0051] The method of ionization using a mass spectrometer is not particularly limited, and may be electrospray ionization (ESI), nano-electrospray ionization (nano-LSI), etc. In this embodiment, the ionization method is preferably ESI.

[0052] Examples of the mass spectrometer include a Fourier transform mass spectrometer, a time-of-flight (Q-TOF) mass spectrometer, a triple quadrupole mass spectrometer, etc. In this embodiment, from the viewpoint of analyzing known components accurately in a short time, the mass spectrometer is preferably a triple quadrupole mass spectrometer.

[0053] In one aspect of this embodiment, the separation step and the analysis step may be performed consecutively. For example, the separation step and the analysis step may be performed consecutively by using a liquid chromatograph-tandem mass spectrometer. The liquid chromatograph-tandem mass spectrometer is typically a liquid chromatograph-triple quadrupole mass spectrometer or a liquid chromatograph-quadrupole / time-of-flight (Q-TOF) mass spectrometer. In such a liquid chromatograph-tandem mass spectrometer, analysis parameters are typically determined to maximize detection sensitivity, i.e., to achieve the best possible conditions, for each component in the eluate separated by the liquid chromatograph.

[0054] In one aspect of this embodiment, it is preferable that the analyzing step further includes quantifying the separated components. The method for quantifying the separated components is not particularly limited, and known methods can be used. Examples of such methods include the absolute calibration curve method and the internal standard method. In another aspect of this embodiment, it is preferable that the analyzing step further includes quantifying the separated components based on a calibration curve prepared in advance.

[0055] <Kit for recovering acidic lipids> A third aspect of this embodiment is a kit for recovering acidic lipids, comprising: a strong anion exchange column; A cleaning solution; an elution solution; Includes:

[0056] The strong anion exchange column, the washing solution, and the elution solution are as described above.

[0057] The kit may further include at least one selected from the group consisting of a microplate, a sample tube, a mobile phase, a bottle containing the mobile phase, and an instruction manual describing the procedure for the recovery method according to the first aspect described above. [Example]

[0058] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0059] <Experiment 1: Method for recovering acidic lipids> (preparation process) Lipid components were extracted from Arabidopsis leaves using the BUME method with chloroform:methanol (volume ratio 1:1) (100 μL) (see Figures 4 and 5). The extracted lipid components (5 μL) were mixed with a mixed solution of 2-propanol and 10 mM ammonium formate (volume ratio 3:1) (100 μL) to prepare a sample (total volume 105 μL). This sample corresponds to the "sample containing acidic lipids."

[0060] (Applicable process) The strong anion exchange column used for solid-phase extraction was an Oasis MAX 30 mg (1 mL cartridge) (Waters). Conditioning was performed by passing 1 mL of methanol through the strong anion exchange column, followed by 500 μL of a 3:1 volumetric ratio mixture of 2-propanol and 10 mM ammonium formate aqueous solution. The sample (total volume 105 μL) prepared in the preparation step above was applied to (passed through) the conditioned strong anion exchange column (see Figures 4 and 5). Alternatively, an InertSep MAX FF 60 mg (3 mL cartridge) could also be used as the strong anion exchange column.

[0061] (Cleaning process) Then, a mixed solution of 2-propanol and 10 mM ammonium formate aqueous solution (volume ratio 3:1) (500 μL to 1 mL) was applied as a washing solution to the strong anion exchange column, and the sample and washing solution that passed through the column were collected as a non-acidic lipid fraction (high peak fraction in Figure 5) (see Figure 5).

[0062] (Recovery process) Then, a mixed solution of 2-propanol:2M ammonium chloride aqueous solution (volume ratio 9:1) (200 μL to 500 μL) was applied to the strong anion exchange column as an extraction solution, and the solution that passed through was collected as an acidic lipid fraction (low peak fraction in Figure 5) (hereinafter, sometimes referred to as "acidic lipid fraction A") (see Figure 5). The collected acidic lipid fraction was dispensed into two containers, and one of them was again subjected to the above-mentioned application and collection steps and collected as an acidic lipid fraction (hereinafter, sometimes referred to as "acidic lipid fraction B").

[0063] <Experiment 2: Method for analyzing acidic lipids in a sample> (Sample acquisition process) The non-acidic lipid fraction, acidic lipid fraction A, and acidic lipid fraction B collected in Experiment 1 were used as analytical samples. Each fraction was dried with nitrogen gas. The non-acidic lipid fraction was dissolved in a 2-propanol:water mixed solvent (volume ratio 3:2) (1 mL), and 3 μL was measured. The acidic lipid fraction A and acidic lipid fraction B were each dissolved in 200 μL, and 10 μL and 0.5 μL of each fraction were measured.

[0064] (separation process, analysis process) The sample before solid-phase extraction (the sample prepared in the preparation step), the analytical sample for the non-acidic lipid fraction, the analytical sample for the acidic lipid fraction A, and the analytical sample for the acidic lipid fraction B were analyzed under the following LC-MS / MS analytical conditions and transitions (see Figures 4 and 5).

[0065] (LC-MS / MS analysis conditions for non-acidic lipid fractions) [Table 1-1]

[0066] [Table 1-2]

[0067] (Non-acidic lipid fraction transitions) [Table 1-3]

[0068] [Table 1-4]

[0069] [Table 1-5]

[0070] (LC-MS / MS analysis conditions for acidic lipid fractions) [Table 2-1]

[0071] [Table 2-2]

[0072] (Transition of acidic lipid fraction) [Table 2-3]

[0073] [Table 2-4]

[0074] [Table 2-5]

[0075] [Table 2-6]

[0076] Based on the results of the above analysis, the recovery rate of the non-acidic lipid fraction was calculated using the following formula: The results are shown in Figure 6. In Figure 6, the horizontal axis represents the detected lipids, and the vertical axis represents the recovery rate. Recovery rate = (peak area in non-acidic lipid fraction) / (peak area in sample before solid phase extraction)

[0077] Furthermore, based on the results of the above analysis, the recovery rate of the acidic lipid fraction was calculated using the following formula: The results are shown in Figure 7. In Figure 7, the horizontal axis represents the detected lipids, and the vertical axis represents the recovery rate. Recovery rate = (peak area in acidic lipid fraction B) / (peak area in acidic lipid fraction A)

[0078] 6, it was found that the lipids contained in the non-acidic lipid fraction were MGDG, DGDG, PC, and PE. Furthermore, the recovery rate of the non-acidic lipid fraction was generally 0.9 to 1.0, which was good.

[0079] From the results in Figure 7, it was found that the lipids contained in the acidic lipid fraction were SQDG, PG, and PI. In addition, the recovery rate of the acidic lipid fraction was generally 0.7 to 0.85, which was good. Furthermore, no lipids other than acidic lipids were detected in the acidic lipid fraction. From the above results, it was demonstrated that the method for recovering acidic lipids according to the examples can recover acidic lipids simply and reliably.

[0080] [Aspect] It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0081] (Section 1) A method for recovering acidic lipids according to one embodiment includes the steps of: preparing a sample containing the acidic lipids; applying the prepared sample to a strong anion exchange column; washing the strong anion exchange column with a wash solution; and recovering the acidic lipids by applying an elution solution to the strong anion exchange column. The method for recovering acidic lipids according to the first aspect provides a simple and reliable method for recovering acidic lipids.

[0082] (Section 2) In the recovery method described in item 1, the stationary phase of the strong anion exchange column has a quaternary ammonium group. According to the recovery method described in item 2, acidic lipids can be recovered more reliably.

[0083] (Section 3) In the recovery method according to item 1 or 2, the acidic lipid comprises at least one selected from the group consisting of phosphatidylglycerol, phosphatidylinositol, sulfoquinovosyldiacylglycerol, and phosphatidic acid. According to the recovery method according to item 3, these acidic lipids can be suitably recovered.

[0084] (Section 4) In the recovery method according to any one of items 1 to 3, the washing solution contains 2-propanol and a volatile salt buffer solution. According to the recovery method according to item 4, the strong anion exchange column can be washed effectively, and lipids other than acidic lipids can be removed from the column.

[0085] (Section 5) In the recovery method according to any one of items 1 to 4, the elution solution contains 2-propanol and ammonium chloride, ammonium sulfate, ammonium citrate, or ammonium nitrate. According to the recovery method according to item 5, acidic lipids can be recovered efficiently.

[0086] (Section 6) In the recovery method according to any one of items 1 to 5, the sample is derived from a plant. According to the recovery method according to item 6, trace amounts of acidic lipids contained in a plant-derived sample can be recovered efficiently.

[0087] (Section 7) The recovery method according to any one of items 1 to 6 further comprises collecting the wash solution passed through the strong anion exchange column in the washing step. The recovery method according to item 7 makes it possible to recover lipids that have not bound to the strong anion exchange column.

[0088] (Section 8) A method for analyzing acidic lipids in a sample according to one embodiment includes a sample obtaining step of obtaining an analytical sample containing the acidic lipids recovered from the sample by the method for recovering acidic lipids according to any one of paragraphs 1 to 7, a separation step of separating the analytical sample into components by liquid chromatography, and an analysis step of analyzing the separated components by a mass spectrometer. The method for analyzing acidic lipids in a sample according to paragraph 8 allows for accurate analysis of acidic lipids contained in the sample.

[0089] (Section 9) A kit for recovering acidic lipids according to one embodiment includes a strong anion exchange column, a washing solution, and an elution solution. The kit described in item 9 provides a simple and reliable method for recovering acidic lipids.

[0090] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0091] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims rather than the above-described embodiments and examples, and it is intended to include any modifications within the scope of the claims that are equivalent to the claims.

Claims

1. A method for recovering acidic lipids, comprising: A preparation step of preparing a sample containing the acidic lipid; applying the prepared sample to a strong anion exchange column; a washing step in which a wash solution is applied to the strong anion exchange column; a recovery step of applying an elution solution to the strong anion exchange column to recover the acidic lipids; A method for recovering acidic lipids, comprising:

2. The method for recovering acidic lipids according to claim 1, wherein the stationary phase of the strong anion exchange column has a quaternary ammonium group.

3. The method for recovering acidic lipids according to claim 1 or claim 2, wherein the acidic lipids include at least one selected from the group consisting of phosphatidylglycerol, phosphatidylinositol, sulfoquinovosyldiacylglycerol, and phosphatidic acid.

4. The method for recovering acidic lipids according to claim 1 or 2, wherein the washing solution contains 2-propanol and a buffer solution of a volatile salt.

5. The method for recovering acidic lipids according to claim 1 or 2, wherein the elution solution contains 2-propanol and ammonium chloride, ammonium sulfate, ammonium citrate, or ammonium nitrate.

6. The method for recovering acidic lipids according to claim 1 or claim 2, wherein the sample is derived from a plant.

7. The method for recovering acidic lipids according to claim 1 or claim 2, further comprising collecting the washing solution that has passed through the strong anion exchange column in the washing step.

8. 1. A method for analyzing acidic lipids in a sample, comprising: A sample obtaining step of obtaining an analytical sample containing the acidic lipid recovered from the sample by the method for recovering acidic lipids according to claim 1 or 2; a separation step of separating the analytical sample into components by liquid chromatography; an analyzing step of analyzing the separated components using a mass spectrometer; A method for analyzing acidic lipids in a sample, comprising:

9. A kit for recovering acidic lipids, comprising: a strong anion exchange column; A cleaning solution; an elution solution; Includes a kit.

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