Plasmalogen detection method and plasmalogen detection kit

Abstract

This plasmalogen detection method comprises: a sample preparation step for mixing a Schiff's reagent and a specimen containing a plasmalogen to obtain a sample; a reaction step for reacting the plasmalogen in the sample with the Schiff's reagent; and a contact step for bringing at least some of the sample reacted in the reaction step into contact with a stationary phase containing a silica gel.

Description

Method for detecting plasmalogen and kit for detecting plasmalogen

[0001] The present invention relates to a method for detecting plasmalogen and a kit for detecting plasmalogen.

[0002] Plasmalogen is a kind of phospholipid having an antioxidant action and is one of glycerophospholipids. Plasmalogen is present in all tissues of mammals and accounts for about 18% of the phospholipids in the human body. Since many plasmalogens are bound to polyunsaturated fatty acids such as docosahexaenoic acid and arachidonic acid, they are involved in the storage of polyunsaturated fatty acids and the release of secondary messengers of intercellular signals such as prostaglandins and leukotrienes produced from these polyunsaturated fatty acids. Furthermore, plasmalogen is involved in cell fusion, ion transport, etc. Since the vinyl ether bond (alkenyl bond) of plasmalogen is particularly sensitive to oxidative stress, plasmalogen also plays an antioxidant function in cells. In vivo, plasmalogen exhibits a protective action against oxidative stress. For example, Non-Patent Documents 1 and 2 report that the concentration of plasmalogen in plasma serves as a biomarker for arteriosclerosis, cancer, etc.

[0003] It is known that plasmalogen is decreased in neurodegenerative diseases such as dementia, Parkinson's disease, depression, and schizophrenia. For example, in Non-Patent Document 3, it was reported that ethanolamine-type plasmalogen is significantly decreased in the frontal lobe and hippocampus in the brain of Alzheimer's disease. Non-Patent Document 4 discloses that the concentration of plasmalogen in the blood of patients with Alzheimer's type dementia is decreased.

[0004] As the relationship between plasmalogen and diseases is elucidated, a technique for easily measuring plasmalogen in vivo is required. It is known that the vinyl ether bond of plasmalogen reacts specifically and quantitatively with iodine in a methanol solution. As shown in Non-Patent Document 5, plasmalogen can be quantified by measuring the decrease in the absorption of light with a wavelength of 355 nm by iodine. The method using iodine disclosed in Non-Patent Document 5 is relatively simple, but has the disadvantage of low detection sensitivity.

[0005] The vinyl ether bond in plasmalogens is unstable in acid and is broken down into aliphatic aldehydes and lysophospholipids by acid. Aliphatic aldehydes can usually be measured as dimethyl acetals using gas chromatography-mass spectrometry. Lysophospholipids, on the other hand, can be measured by two-dimensional thin-layer chromatography, etc.

[0006] Patent Document 1 discloses a method for quantifying ethanolamine-type plasmalogens, which includes a step of treating ethanolamine-type lysoplasmalogen, produced by hydrolyzing ethanolamine-type plasmalogens in a sample with phospholipase A1, with phospholipase D.

[0007] Patent Document 2 discloses a method for quantifying choline-type plasmalogens that utilizes the action of hydrolyzing the phosphate ester at the sn-3 position of choline-type plasmalogens using phospholipase D, which acts on choline-type plasmalogens, thereby releasing choline.

[0008] Furthermore, by using liquid chromatography-mass spectrometry (LC-MS), it is possible to accurately identify lipids in a mixture and identify plasmalogens by detecting characteristic fragment ions of fatty acids or alcohols esterified with the head group class and glycerol backbone.

[0009] Non-patent document 6 qualitatively analyzes the presence or absence of plasmalogens in Clostridium perfringens by a color reaction between aliphatic aldehydes produced by acid treatment of plasmalogens and Schiff's reagent (fuchsinaldehyde reagent).

[0010] Japanese Patent Publication No. 2016-111929 Japanese Patent Publication No. 2014-082991

[0011] Nishimukai Megumi, et al., "Absorption characteristics of plasmalogens and usefulness of blood plasmalogens as biomarkers," Oleoscience, 2015, Vol. 15, No. 2, 53-60. Marcia Cristina Fernandes Messias, et al., "Plasmalogen lipids: functional mechanism and their involvement in gastrointestinal cancer," Lipids in Health and Disease, 2018, 17, 41. Zhizhong Guan, et al., "Decrease and Structural Modifications of Phosphatidylethanolamine Plasmalogen in the Brain with Alzheimer Disease”, J Neuropathol Exp Neurol, 1999, 58(7), 740-747 Dayan B. Goodenowe, 17 others, “Peripheral ethanolamine plasmalogen deficiency: a logical causative factor in Alzheimer's disease and dementia”, 2007, J Lipid Res, 48(11), 2485-2498Eugene L. Gottfried and Maurice M. Rapport, “The biochemistry of plasmalogens. I. Isolation and characterization of phosphotidal choline, a pure native "plasmalogen", 1962, J Biol Chem, 237, 329-333 David R. Jackson, and 7 others, “Plasmalogen Biosynthesis by Anaerobic Bacteria: Identification of a Two-Gene Operan Responsible for Plasmalogen Production in Clostridium perfringens”, 2021, ACS Chem Biol, 16, 6-13

[0012] All of the methods disclosed in Non-Patent Document 5, Patent Documents 1 and 2 require an extraction process of the lipid fraction using an organic solvent. These methods, or detection of plasmalogens by gas chromatography or liquid chromatography-tandem mass spectrometry (LC-MS / MS), require time-consuming and expensive analytical equipment, making it difficult to measure a large number of samples. Furthermore, the method using Schiff's reagent described in Non-Patent Document 6 requires microscopic observation of cell coloration, which is time-consuming and difficult to apply to a large number of samples.

[0013] This invention has been made in view of the above circumstances, and aims to provide a method for detecting plasmalogens and a kit for detecting plasmalogens that can detect plasmalogens simply and quickly.

[0014] A method for detecting plasmalogens according to a first aspect of the present invention includes: a sample preparation step of mixing a sample containing plasmalogens with a Schiff reagent to obtain a sample; a reaction step of reacting the plasmalogens in the sample with the Schiff reagent; and a contact step of contacting at least a portion of the sample reacted in the reaction step with a stationary phase containing silica gel.

[0015] The sample is plasma, and the reaction step and the contact step further include a separation step of centrifuging the sample, wherein in the contact step, at least a portion of the supernatant of the sample is brought into contact with the stationary phase.

[0016] The solvent for the Schiff reagent may be aqueous sulfite.

[0017] A kit for detecting plasmalogens according to a second aspect of the present invention comprises a stationary phase containing silica gel into which a sample containing a plasmalogen is brought into contact with a sample mixed with Schiff's reagent.

[0018] The plasmalogen detection kit according to the second aspect of the present invention may further comprise the Schiff reagent.

[0019] According to the present invention, plasmalogens can be detected simply and quickly without the need for lipid fraction extraction.

[0020] This figure shows the configuration of the detection kit according to Embodiment 2 of the present invention. This figure shows the separation method of plasmalogen by high-performance liquid chromatography (HPLC) according to Example 1. This figure shows the chromatogram of the lipid fraction extracted by HPLC in Example 1. This figure shows the chromatogram of ethanolamine-type plasmalogen (PL-PE) in Example 1. This figure shows an image of the area on the silica gel TLC plate where the sample was dropped according to Example 1. This figure shows an image of the paper for paper chromatography on which the sample according to Comparative Example 1 was dropped. This figure shows an image of the area on the silica gel TLC plate where the sample was dropped according to Example 2.

[0021] Embodiments of the present invention will be described with reference to the drawings. However, the present invention is not limited to the embodiments and drawings described below. In the embodiments described below, expressions such as “having,” “including,” or “containing” also include the meaning of “consisting of” or “composed of.”

[0022] (Embodiment 1) The plasmalogen detection method according to this embodiment includes a sample preparation step, a reaction step, and a contact step. In the sample preparation step, a sample containing plasmalogen is mixed with Schiff's reagent to obtain a sample. Note that the sample may not contain plasmalogen, in which case the detection method will indicate that the sample does not contain plasmalogen or that it is below the detection limit. Preferably, in the sample preparation step, Schiff's reagent and the sample are mixed under acidic conditions. For example, if the solvent of Schiff's reagent is acidic, the Schiff's reagent and the sample can be mixed under acidic conditions. More specifically, the pH of the mixture obtained by mixing Schiff's reagent and the sample should be less than 7. Preferably, the pH is less than 5, less than 4, less than 3, or less than 2. The solvent of Schiff's reagent is preferably sulfurous acid.

[0023] The specimen is not particularly limited and may be a living organism's bodily fluid, cell culture medium, etc. Examples of specimens include blood, plasma, serum, urine, sweat, cerebrospinal fluid, bile, pancreatic juice, amniotic fluid, ascites, etc. Preferably, the specimen does not contain cells. Preferably, the specimen is plasma. The living organism is a human or an animal other than a human, preferably a mammal. Mammals include, for example, primates such as chimpanzees, laboratory animals such as rats, mice, and rabbits, domestic animals such as pigs, cows, horses, sheep, and goats, and companion animals such as dogs and cats.

[0024] Schiff's reagent is a reagent that exhibits color specifically to aldehydes. "Exhibiting color specifically to aldehydes" means that it reacts with aldehydes and not with ketones to produce color. Because Schiff's reagent does not react with ketones, it reacts specifically with plasmalogens even if the sample contains proteins. Schiff's reagent is not particularly limited as long as it exhibits color specifically to aldehydes, and may be, for example, fuchsin, fuchsin sulfite reagent, or pararoseaniline-based reagent. Schiff's reagent may be a commercially available product, or it may be prepared by dissolving basic fuchsin in hot water, cooling it, adding anhydrous sodium sulfite and concentrated hydrochloric acid, and then diluting it with water as appropriate. The concentration of the component that reacts with aldehydes in the Schiff's reagent mixed with the sample is not particularly limited, but for example, it may be 0.1-1%, 0.05-0.5%, 0.03-0.3%, or 0.02-0.2%.

[0025] The mixing ratio of the sample and Schiff reagent mixed in the sample preparation step is not particularly limited. For example, the volume ratio of the sample to Schiff reagent (sample:Schiff reagent) may be 1:1 to 1:20, 1:1.5 to 1:10, 1:1.8 to 1:8, or 1:2 to 1:5. In addition, in the sample preparation step, the sample may be diluted with a solvent such as ethanol before being mixed with the Schiff reagent.

[0026] In addition, during the sample preparation step, a control sample may be obtained by mixing the solvent of a sample that does not contain plasmalogens, for example, a sample that contains plasmalogens, with Schiff's reagent. For example, when a sample such as plasma is diluted with ethanol as a solvent, ethanol and Schiff's reagent are mixed. The mixing ratio of the solvent and Schiff's reagent mixed in the preparation of the control sample is not particularly limited and is, for example, the same as the mixing ratio of the sample and Schiff's reagent in the sample preparation step.

[0027] In the reaction step, the plasmalogen in the sample is reacted with Schiff's reagent. Here, the aldehyde generated from the vinyl ether bond of the plasmalogen is reacted with Schiff's reagent. To react the aldehyde with Schiff's reagent, for example, the sample can be incubated at room temperature, more preferably 37°C. The reaction time is, for example, 5 to 60 minutes, 10 to 50 minutes, 20 to 40 minutes, or 25 to 35 minutes. When using a control sample, in the reaction step, the control sample can be reacted under the same conditions as the sample containing the plasmalogen and Schiff's reagent.

[0028] In the contact step, at least a portion of the sample reacted in the reaction step is brought into contact with a stationary phase containing silica gel. The stationary phase containing silica gel is not particularly limited as long as contact with the sample is maintained, and is, for example, a thin layer formed of silica gel. The thickness of the stationary phase is not particularly limited, but is, for example, 50 to 1000 μm, 100 to 800 μm, 150 to 500 μm, or 180 to 250 μm. The average pore size of the silica gel is not particularly limited, but is, for example, 10 to 100 Å, 20 to 90 Å, 30 to 80 Å, 40 to 70 Å, or 50 to 70 Å. Particularly preferably, the average pore size of the silica gel is 60 Å. Preferably, the stationary phase is supported on the surface of a support. The support is not particularly limited, and is, for example, an aluminum sheet, glass, paper, etc. Preferably, in the contact step, a thin-layer chromatography plate (silica gel plate) using silica gel as the stationary phase (support) is used.

[0029] In the contact step, the sample only needs to be maintained in contact with the stationary phase, and the manner of contact between the sample and the stationary phase is arbitrary. For example, in the contact step, the sample is placed on a silica gel plate by being dropped onto it. The volume of the sample placed on the silica gel plate is, for example, 2-50 μL, 5-40 μL, 8-30 μL, or 10-20 μL.

[0030] If the sample is plasma, the detection method according to this embodiment may further include a separation step of centrifuging the sample between the reaction step and the contact step. In this case, in the contact step, at least a portion of the supernatant of the sample may be brought into contact with the stationary phase.

[0031] The detection method according to this embodiment may further include a drying step after the contact step, in which the sample in contact with the stationary phase is dried. The sample is dried by any method that does not affect the diffusion rate of the unreacted Schiff reagent and the reaction product between the plasmalogen and the Schiff reagent. By reducing the volume of the sample in contact with the stationary phase, it can be dried in a short time even by natural drying.

[0032] As shown in Examples 1 and 2 below, the unreacted Schiff reagent diffuses more rapidly in the silica gel plate than the reaction product of plasmalogen and Schiff reagent, allowing the reaction product to be visually identified at a different location from the unreacted Schiff reagent. According to the detection method of this embodiment, plasmalogen can be detected simply and quickly without the need for lipid fraction extraction.

[0033] Furthermore, in the contact step, the sample may be brought into contact with multiple locations on the stationary phase. This allows for rapid determination of whether or not multiple samples contain plasmalogens. Alternatively, in the contact step, the sample may be brought into contact with at least one location on the stationary phase supported by each of multiple supports. In addition, the stationary phase after the contact step may be imaged with a camera, and the reaction product of the plasmalogen and Schiff reagent may be detected by analyzing the obtained image. This allows for high-throughput processing of a large number of samples.

[0034] (Embodiment 2) The plasmalogen detection kit 10 according to this embodiment will be described with reference to Figure 1. Unless otherwise specified, the description of the detection method according to Embodiment 1 above can be used to describe the plasmalogen detection kit 10 according to this embodiment. The plasmalogen detection kit 10 may be used in the detection method according to Embodiment 1 above, although it is not particularly limited.

[0035] As shown in Figure 1, the detection kit 10 comprises a silica gel plate 1, a substrate 2 that holds the silica gel plate 1 on its upper surface, and a cover 3 that is placed over the substrate 2 with the silica gel plate 1 interposed therebetween. The silica gel plate 1 has a stationary phase 5 containing silica gel supported on the surface of an aluminum sheet 4 which serves as a support.

[0036] The cover 3 has through-holes 6 at positions corresponding to the stationary phase 5 when the cover 3 is placed over the substrate 2. The stationary phase 5 is brought into contact with a sample containing plasmalogen and a Schiff reagent mixture. More specifically, when the user drops the sample into the through-holes 6, the sample is placed on and comes into contact with the stationary phase 5.

[0037] When a sample containing plasmalogen is placed on the stationary phase 5, the unreacted Schiff reagent diffuses from the position where the sample is placed on the stationary phase 5 and diffuses into the area indicated by the diffusion position 7. On the other hand, the reaction product of plasmalogen and Schiff reagent, which has a lower diffusion rate than the unreacted Schiff reagent, diffuses into the area indicated by the range 8, which is located inside the diffusion position 7, centered on the position where the sample is placed on the stationary phase 5. Preferably, the size of the through-hole 6 is set such that, when the volume of the sample placed on the stationary phase 5 is within a predetermined range, the range 8 is visible when looking at the stationary phase 5 from the through-hole 6, but the diffusion position 7 is not visible. In this way, the user can determine whether or not the sample contains plasmalogen by checking for discoloration in the range 8 from the through-hole 6, for example, the presence or absence of reddish-purple when the stationary phase 5 is white.

[0038] According to the detection kit 10 of this embodiment, plasmalogens can be detected simply and quickly.

[0039] The plasmalogen detection kit according to this embodiment may further include a Schiff reagent. Furthermore, although the detection kit 10 comprises a substrate 2 and a cover 3, it may also include at least a silica gel plate 1.

[0040] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

[0041] [Example 1: Detection of Plasmalogen Using Schiff's Reagent] 1. Preparation of sPls Plasmalogen (sPls) consisting only of PL-PE was prepared from scallops (Mizuhopecten yessoensis) by the following method. sPls was extracted from raw scallop adductor muscles using hexane, as described in Japanese Patent No. 7304643. The steps for extracting plasmalogen from scallops are as follows: (1) Cokurase P (manufactured by Mitsubishi Chemical Foods) and phospholipase A1 (PLA1, manufactured by Mitsubishi Chemical Foods) were added to the scallops and decomposed for 1 hour. (2) Next, hexane / isopropanol was added and stirred, and after standing, the supernatant was filtered by suction. (3) Sodium sulfate aqueous solution was added to the filtrate and mixed well. (4) The upper layer was dried using a rotary evaporator. (5) Add 40 mL of acetone cooled to 4°C and mix. (6) Separation was performed using a centrifuge at 3000 rpm for 10 minutes at 4°C. (7) The supernatant was discarded and the precipitate was collected. (8) It was dried overnight in a desiccator. Separation was performed by HPLC using the mobile phase solvent shown in Figure 2A, following the specifications of Japanese Patent No. 5430566. Figure 2B shows the lipid composition of the fraction extracted in steps (1) to (8) above. As shown in Figure 2C, only PL-PE was separated and designated as sPlus.

[0042] 2. 18.5 μL of Schiff solution obtained by diluting a plasmaogen detection Schiff reagent (pH < 2.0, manufactured by Sigma, 84655) 5-fold with sulfurous acid water was mixed with 1.5 μL of sPls at 1 mg / mL or 1.5 μL of ethanol to prepare a sample. The sample was reacted at 37 °C for 30 minutes. 10 μL of the sample was dropped onto a silica gel TLC plate (manufactured by AS ONE, model number: A1020, product number: 4-2547-05). The silica gel TLC plate was dried for about 5 minutes, and the dropped portion of the silica gel TLC plate was photographed with a scanner.

[0043] 3. As shown in Fig. 3, at the location where the sample containing sPls was dropped, the reaction product of sPls and the Schiff reagent could be visually confirmed inside the ring colored with the Schiff reagent with a large diffusion rate that did not react with sPls. At the location where the sample containing ethanol instead of sPls was dropped, the reaction product could not be confirmed inside the ring.

[0044] [Comparative Example 1: Detection of Plasmaogen Using Schiff Reagent] The sample prepared in Example 1 above was dropped onto paper for paper chromatography. As shown in Fig. 4, both the sample containing sPls and the sample not containing sPls were colored in the same way and could not be distinguished.

[0045] [Example 2: Detection of Plasmaogen in Human Plasma Using Schiff Reagent] 1. Detection of Plasmaogen Venous blood from subjects A to H was collected in heparin-containing blood collection tubes (manufactured by Terumo), centrifuged at 1000 × g for 5 minutes, and the supernatant plasma was collected and stored frozen. 7 μL of ethanol was added to 3 μL of plasma, and further 30 μL of the same Schiff solution as in Example 1 above was added to obtain a sample. The sample was reacted at 37 °C for 30 minutes and centrifuged for 2 minutes (10,000 rpm). 15 μL of the supernatant of the sample was dropped onto a silica gel TLC plate (manufactured by AS ONE, model number: A1020, product number: 4-2547-05). The silica gel TLC plate was dried for about 5 minutes, and the dropped portion of the silica gel TLC plate was photographed with a scanner.

[0046] 2. Quantification of Plasma Lipoproteins in Human Plasma by LC-MS (1) Preparation of Stock Standard Solution As reagents, phosphatidylethanolamine (PE, manufactured by Funakoshi, A-34, dissolved in chloroform at 10 mg / mL), phosphatidylcholine (PC, manufactured by Funakoshi, A-29B, dissolved in chloroform at 5 mg / mL), and sphingomyelin (SM, manufactured by Funakoshi, A-401) were used. 25 mg of SM was prepared in chloroform at 5 mg / mL.

[0047] 100 μL of PE, 200 μL each of PC and SM were taken into separate screw-top tubes.

[0050] , They were dried under nitrogen gas, and 1 mL each of isopropanol / chloroform (2:1) was added to PE, PC, and SM and adjusted to 1 mg / mL. After mixing, they were sonicated and stored at -30 °C as stock standard solutions.

[0048] (2) Preparation of Standard Solutions for Measurement The stock standard solutions were diluted with HIP (3:2) on ice (5, 10, 15, 20, 25, 50 μg / mL for PE and PC, 20, 40, 60, 80, 100, 200 μg / mL for SM), and each was filtered through a 0.45 μL filter to obtain standard solutions.

[0049] (3) Lipid Extraction from Blood Venous blood from subjects A to H was collected in heparinized blood collection tubes (manufactured by Terumo), centrifuged at 1000 × g for 5 minutes, and the supernatant plasma was recovered and stored frozen. The stored plasma was thawed by immersing it in room temperature water and centrifuged at 13,000 rpm for 5 minutes. The plasma was pipetted with a 1 mL pipette and 280 μL was placed in an eppendorf tube. A PLA1 (×2) solution prepared by mixing Phospholipase A1 (manufactured by Sigma) and 0.1 M citrate buffer (HCl pH 4.5) at 1:1 was added to the above eppendorf tube and mixed. The reaction was carried out at 45 °C for 1 hour, and immediately after the reaction, it was immersed in ice water to stop the reaction. After mixing, the plasma treated with PLA1 was pipetted and 100 μL portions were placed in eppendorf tubes.

[0050] Lipids were extracted using the hexane-isopropanol (HIP) method as follows: 800 μL of HIP (3:2) was added to the plasma and mixed. The mixture was treated with ultrasound for 5 minutes, and Na ２ SO ４ 400 μL of water (1 g / 15 mL) was added and mixed. After standing for 5 minutes, 400 μL of the upper layer was taken without pipetting and transferred to another Eppendorf tube. 400 μL of HIP (7:2) was then added and mixed. After standing for 5 minutes, 300 μL of the upper layer was taken without pipetting and combined with the upper layer previously collected in the Eppendorf tube to form the sample. The sample was stored at -30°C until measurement.

[0051] (4) Before LC-MS measurement, the sample is N ２ The sample was dried using gas. The sample was redissolved in 200 μL of HIP (3:2) and filtered through a 0.45 μL filter.

[0052] The prepared standard solutions and samples were each placed in a glass insert with legs, and 10 μL was injected into the LC-MS. The LC-MS conditions are as follows: Column: CHEMCOBOND 5OH(Diol) 3.0×30, 30×250(w) (Chemcoplus) Column temperature: 50°C Ionization mode: API-ES Polarity: Negative Scan Mass range low: 680 Scan Mass range high: 950 Gas temperature: 300°C Drying gas flow: 10.0 L / min Nebulizer Pressure: 45 psig Vcap (negative): 3500 V Solvent A: 53 mM formate-acetonitrile Solvent B: 60 mM ammonium formate and 53 mM formate-water

[0053] Table 1 shows the LC-MS time schedule.

[0054]

[0055] 3. Results As shown in Figure 5, in the areas where the sample containing plasmalogen was dropped, the reaction product of sPls and Schiff's reagent was visible within a ring colored with unreacted Schiff's reagent, at a concentration corresponding to the plasmalogen concentration quantified by LC-MS.

[0056] This invention allows for various embodiments and modifications without departing from the broad spirit and scope of the invention. Furthermore, the embodiments described above are for illustrative purposes only and do not limit the scope of the invention. In other words, the scope of the invention is indicated not by the embodiments, but by the claims. Various modifications made within the scope of the claims and the equivalent scope of the meaning of the invention are considered to be within the scope of the invention.

[0057] This application is based on Japanese Patent Application No. 2024-220479, filed on 17 December 2024. The entire specification, claims, and drawings of Japanese Patent Application No. 2024-220479 are incorporated herein by reference.

[0058] This invention is useful for detecting plasmalogens.

[0059] 1. Silica gel plate 2. Substrate 3. Cover 4. Aluminum sheet 5. Stationary phase 6. Through hole 7. Diffusion position 8. Range 10. Detection kit

Claims

1. A method for detecting plasmalogens, comprising: a sample preparation step of mixing a sample containing a plasmalogen with a Schiff reagent to obtain a sample; a reaction step of reacting the plasmalogen in the sample with the Schiff reagent; and a contact step of contacting at least a portion of the sample reacted in the reaction step with a stationary phase containing silica gel.

2. The method for detecting plasmalogens according to claim 1, wherein the sample is plasma, and further comprises a separation step of centrifuging the sample between the reaction step and the contact step, and in the contact step, at least a portion of the supernatant of the sample is brought into contact with the stationary phase.

3. The method for detecting plasmalogens according to claim 1 or 2, wherein the solvent of the Schiff reagent is sulfurous acid.

4. A plasmalogen detection kit comprising a stationary phase containing silica gel for contacting a sample containing plasmalogens with a sample mixed with Schiff's reagent.

5. The plasmalogen detection kit according to claim 4, further comprising the Schiff reagent.

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