KR102118067B1 - Extraction method of lipids using spin column with super absorbent polymer beads - Google Patents

Abstract

The present invention relates to a method for extracting lipids using a spin column filled with super absorbent polymer beads (SAPB). The method for extracting lipids according to the present invention is more sensitive than the conventional Folch method and SAP method. It has the advantage of excellent reproducibility and quantification.

Description

Extraction method of lipids using spin column filled with super absorbent polymer beads {EXTRACTION METHOD OF LIPIDS USING SPIN COLUMN WITH SUPER ABSORBENT POLYMER BEADS}

The present invention relates to a method for extracting lipids using a spin column filled with super absorbent polymer beads (SAPB).

Lipids are major components of biological cell membranes and are involved in a number of biochemical functions and metabolic processes such as intracellular cellular signaling, secretion and energy storage. In addition, lipids are known to play an important role in biological mechanisms and are closely related to diseases such as diabetes, Alzheimer's disease, arteriosclerosis, and cancer, which has led to extensive and quantitative studies of lipids in bioanalytical chemistry. Is being made. The most important issue in the analysis of a sample is the loss of the sample due to the sample processing process. To overcome this, methods have been developed to reduce sample delivery and loss volume during sample preparation. In addition, the types of lipids vary according to the polarity of the lipids and the molecular skeleton structure, which complicates simultaneous analysis of all types of lipids with a high recovery rate. Despite highly sophisticated lipid analysis methods, successful analysis is determined by efficient extraction and separation methods of complex lipid mixtures from biological samples such as cells, tissues, and body fluids.

Traditional lipid extraction methods include the Folch (or modified Folch) method or the Bligh/Dyer method method based on the liquid-liquid extraction (LLE) method. In general, the LLE method is not suitable for the analysis of a small amount of sample and takes a long time to extract lipids and also shows a low recovery rate. In particular, when extracting and analyzing lipids from biological fluid samples such as plasma, urine or body fluids, the removal of moisture is a prerequisite. On the other hand, Patent No. 10-1816288 relates to a method for pre-treatment of a sample using a super absorbent polymer and a device therefor (hereinafter also referred to as a “SAP” method), which uses a super absorbent polymer powder and lipids without requiring a centrifugation step. Although a simplified method of extraction has been disclosed, there is a limitation in separating organic layers quantitatively by centrifugation in a typical Eppendorf tube, and there is a problem in that convenience, reproducibility, and quantification are poor.

Accordingly, the present inventors are using a spin column filled with super absorbent polymer beads (SAPB) while researching to improve the procedure of the existing Folch and SAP methods used to extract lipids and increase separation efficiency. In the case of centrifuging the dissolved lipids by adding an organic solvent after the aqueous solution in the sample has been exchanged with SAPB, it is confirmed that the lipids can be separated with high sensitivity, reproducibility and quantification, and the present invention (Modified SAP method) Was completed.

An object of the present invention is to provide a method for extracting lipids using a spin column filled with super absorbent polymer beads (SAPB).

As used herein, the term “Super Absorbent Polymer (SAP)” refers to a synthetic polymer material having the ability to absorb moisture up to 500 to 1,000 times its own weight.

As used herein, the term “sample” refers to a compound or composition to be analyzed containing an analyte, and a sample that can be used in the present invention is an aqueous phase or a similar flowable material including an aqueous solution. The sample is a biological sample, and includes bio-derived body components such as whole blood, plasma, serum, urine, saliva, manure, lymph fluid, amniotic fluid, and other fluid materials including liquid extracted from tissues, cells, and microorganisms. do.

The principle of extracting lipids using the superabsorbent polymer beads according to the present invention is shown in FIG. 1. The present invention is a conventional modified by using a spin column filled with super absorbent polymer beads (SAPB) to separate the lipid dissolved in the organic solvent added to the added organic solvent through the interaction with SAPB through centrifugation. Samples in a small amount (for example, 10 μL or less in the case of plasma) can be extracted quickly and with high sensitivity and reproducibility compared to the Folch method and the SAP method.

The present invention is to provide a method for extracting lipids using a spin-colum filled with super absorbent polymer beads (SAPB), the method comprising:

(A) adding a water-soluble sample to a spin-column filled with a super absorbent polymer bead to absorb the mixture;

(B) An aqueous solvent layer in which the organic solvent liquid layer is solidified by separately dissolving an aqueous layer in which a water-soluble substance in a water-soluble sample is absorbed into a super-absorbent resin bead and a hydrophobic substance in an organic solvent layer by adding an organic solvent to the superabsorbent polymer beads And separated from; And

(C) is characterized in that it comprises a step of separating the organic solvent layer containing the lipid by centrifugation from the aqueous layer containing the superabsorbent polymer beads.

In the method for extracting lipids according to the present invention, the average diameter of the beads is 1.5 mm or less.

In the method of extracting lipids according to the present invention, the sample is characterized in that it is selected from the group consisting of whole blood, plasma, serum, urine, saliva, manure, lymph fluid, amniotic fluid, tissue, cells and microorganisms.

In the method of extracting the lipid according to the present invention, the organic solvent is an alkyl ether (for example, diisopropyl ether, methyl t-butyl ether (MTBE), ethyl t-butyl ether, methyl t-pentyl ether (TAME) ), ethyl t-pentyl ether, tetrahydrofuran (THF), 1,4-dioxane, ethylene glycol dimethyl ether or diethyl ether, diethylene glycol dimethyl ether or diethyl ether); Dialkyl ketones (e.g. methyl ethyl ketone, methyl i-propyl ketone or methyl i-butyl ketone, nitriles (e.g. acetonitrile, propionitrile, butylonitrile or benzonitrile)) amides (e.g. , N,N-dimethyl-formamide (DMF), N,N-dimethyl-acetamide, N-methyl-formanilide, N-methyl-pyrrolidone or hexamethyl-phosphoramide); esters (e.g. , Methyl, ethyl, n- or i-propyl, n-, i- or s-butyl acetate) alcohols (e.g. ethanol, n- or i-propanol, n-, i-, s- or t- Butanol) and sulfoxide.

In the method for extracting a lipid according to the present invention, the method includes: (A) adding a water-soluble sample to a spin-colum filled with a superabsorbent polymer bead to absorb the mixture; (B) By adding MeOH and MTBE (methyl tert-butyl ether) to the superabsorbent polymer beads, the aqueous layer and the hydrophobic material in which the water-soluble substance in the water-soluble sample is absorbed into the superabsorbent polymer beads are separately dissolved and dissolved in an organic solvent layer, respectively. Separating the solvent liquid layer from the solidified aqueous layer; And (C) separating the organic solvent layer containing the lipid by centrifuging for 1 minute at 6,000 rpm from the aqueous solution layer containing the superabsorbent polymer beads.

According to the method for extracting lipids using the superabsorbent polymer beads of the present invention, it is possible to extract lipids from a small amount of samples for a short time compared to the conventional Folch method using the superabsorbent polymer beads, and also compared with the conventional SAP method. It has the advantage of excellent reproducibility, sensitivity and quantification. Therefore, the present invention is expected to have a very high industrial applicability in the biomedical and chemical fields, such as the development of a sample pre-treatment diagnostic kit capable of extracting and concentrating disease-related biomarkers in biological fluids necessary for disease diagnosis.

1 shows the principle of extracting lipids using the superabsorbent polymer beads according to the present invention.
Figure 2 shows the results of the recovery test (recovery test) according to the extraction method of Examples 1 and 3
Figure 3 shows the sensitivity and quantitative comparison results in the cation mode according to the extraction method of Examples 1 to 3.
Figure 4 shows the results of comparing the sensitivity and quantitative in the anion mode according to the extraction method of Examples 1 to 3.
Figure 5 shows the results of the reproducibility comparison in the cation mode according to the extraction method of Examples 1 to 3.
6 shows a comparison result of reproducibility in anion mode according to the extraction methods of Examples 1 to 3.

Hereinafter, various examples are presented to help understanding of the invention. The following examples are provided only for easier understanding of the invention, and the protection scope of the invention is not limited to the following examples.

< Example >

Example One. Folch According to the method Lipids extraction

Plasma was placed in an Eppendorf tube and 200 μL of MeOH and 600 μL of MTBE were continuously added. After vortexing for 30 minutes, centrifugation was performed at 13,000 rpm for 10 minutes. The supernatant was collected and transferred to a new Eppendorf tube, then 200 μL of DW was added. Then, after vortexing for 30 minutes, centrifugation was performed at 13,000 rpm for 10 minutes. The supernatant was collected and transferred to a new Eppendorf tube and dried using nitrogen gas.

Example  2. According to SAP method Lipids extraction

2-15 mg of superabsorbent polymer powder purchased from LG Chemicals, lnc was placed in an Eppendorf tube and plasma samples were added. After the superabsorbent polymer powder was sufficiently swelling, 100 μL of MeOH and 200 μL of MTBE were continuously added. After vortexing for 5 minutes, centrifugation was performed at 13,000 rpm for 10 minutes. The supernatant was collected and transferred to a new Eppendorf tube and dried using nitrogen gas.

Example 3. According to the present invention (modified SAP) Lipids extraction

15 superabsorbent resin beads were placed in a reservoir of a spin column, and plasma samples were added. After the superabsorbent polymer beads were sufficiently swelled, 100 μL of MeOH and 200 μL of MTBE were continuously added. Then, centrifugation was performed at 6,000 rpm for 1 minute. The spin down solvent (organic layer) was transferred to a new Eppendorf tube and dried using nitrogen gas.

< Experimental Example >

Experimental Example  1. Comparison of recovery test results according to extraction method

Lipid standard materials (LPC 17:0, Cer d17:0, SM d17:0, TG 15:0/15:0/15:0, PC to compare the extraction recovery rate of lipids according to Examples 1 to 3) 19:0/19:0, PG 17:0/17:0, PE 17:0/!7:0, PS 17:0/17:0) in the positive and negative modes, respectively, recovery test Proceeded. It was confirmed that the method according to the present invention (Example 3) had a higher recovery rate than Example 1 for all lipid standard substances except TG in the cation mode and all lipid substances in the anion mode.

Experimental Example 2. Sensitivity according to the extraction method and Quantification  compare

LOD test using PC(17:0/17:0) in cation mode and PG(17:0/17:0) in anion mode to compare extraction efficiency of lipids according to Examples 1 to 3, respectively. Proceeded. At this time, the LOD was calculated using 3.3 X standard deviation/slope, and the results are shown in FIGS. 3 and 4.

In the case of cation mode, the LODs of Examples 1, 2 and 3 were 0.2431, 0.3096 and 0.0351 pmole, respectively, 7 times compared to mFolch and 9 compared to SAP when extracting lipids by the method according to the present invention. It was confirmed that the extraction efficiency increased more than twice (FIG. 3 ). In the case of anion mode, the LODs of Examples 1, 2, and 3 were 0.2340, 0.2359, and 0.1280 pmole, respectively, and when extracting lipids by the method according to the present invention, they were extracted twice or more compared to mFolch and SAP. It was confirmed that the efficiency increased (Fig. 4). On the other hand, of comparing the correlation coefficients (r ²⁾ for the linearity shown in the concentration region within (0.5 ~ 40 pmol) of the irradiated standard lipid material for cation mode, Example 1, Example 2 and Example 3 r ² The values were 0.9955, 0.9955, and 0.9997, respectively, and it was confirmed that the case of extracting lipids by the method according to the present invention was excellent in quantitation (FIG. 3). In the case of the anion mode, the r ² values of Examples 1, 2, and 3 were 0.996, 0.999, and 0.9999, respectively, and it was confirmed that the case of extracting lipids by the method according to the present invention was excellent in quantification. (Figure 4).

Experimental Example 3. Comparison of reproducibility according to extraction method

In order to compare stability and reproducibility during lipid extraction according to Examples 1 to 3, an inter-day and intra-day RSD test was performed for a cation mode and an anion mode. At this time, each experiment was conducted 5 times 5 times per day, and the results are shown in FIGS. 5 and 6.

In the case of the cation mode, the inter-day RSD of Example 1, Example 2 and Example 3 was 11.06, 16.35 and 4.96%, respectively, and it was confirmed that the reproducibility was the best when the lipid was extracted by the method according to the present invention. (Figure 5). In the case of anion mode, Inter-day RSD of Example 1, Example 2 and Example 3 was 5.60, 11.00 and 4.73%, respectively, and it was confirmed that the reproducibility was the best when extracting the lipids by the method according to the present invention. Became (Figure 6).

So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

Claims (5)

A method of extracting lipids using a spin-colum filled with super absorbent polymer beads (SAPB), the method comprising:
(A) adding a water-soluble sample to a spin-colum filled with a super absorbent polymer bead to absorb and mix;
(B) by adding an organic solvent to the superabsorbent polymer beads, the aqueous layer in which the water-soluble substance in the water-soluble sample is absorbed into the superabsorbent polymer and the hydrophobic substance are separately dissolved into an organic solvent layer, and the organic solvent liquid layer is solidified with the aqueous layer Separate step; And
(C) comprising the step of separating the organic solvent layer containing a lipid by centrifugation from the aqueous solution layer containing the superabsorbent polymer beads,
The method, characterized in that the average diameter of the superabsorbent polymer beads is 1.5mm or less.
delete According to claim 1,
The sample is characterized in that it is selected from the group consisting of whole blood, plasma, serum, urine, saliva, manure, lymph fluid, amniotic fluid, tissue, cells and microorganisms.
According to claim 1,
The organic solvent is characterized in that one or more selected from the group consisting of alkyl ethers, dialkyl ketones, amides, esters, alcohols and sulfoxides.
According to claim 1,
The method includes: (A) adding a water-soluble sample to a spin-colum filled with a super absorbent polymer bead to absorb the mixture; (B) By adding MeOH and MTBE (methyl tert-butyl ether) to the superabsorbent polymer beads, the aqueous layer and the hydrophobic material in which the water-soluble substance in the water-soluble sample is absorbed by the superabsorbent polymer beads are separated and dissolved into organic solvent layers, respectively Separating the solvent liquid layer from the solidified aqueous layer; And (C) separating the organic solvent layer containing the lipid by centrifuging for 1 minute at 6,000 rpm from the aqueous solution layer containing the superabsorbent polymer beads.

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