A method and device for rapid detection of fatty acid components of trace lipid substances

Abstract

The present invention discloses a method and device for rapidly detecting fatty acid components of trace lipid substances. The device comprises a liner tube and a sample bottle, and the liner tube can be placed in the sample bottle. In the method for rapidly detecting fatty acid components of trace lipid substances using the device, firstly, the trace oil to be tested and the methyl esterification mixed solution are added to the liner tube for reaction, and then the reaction completed solution is transferred to another clean empty sample bottle, and NaCl solution and n-hexane solution are added to vortex centrifuge to extract the supernatant into another clean liner tube, and finally the supernatant in the liner tube is concentrated by nitrogen blowing, and then the liner tube is placed in the sample bottle for gas chromatograph to detect the fatty acid components. The present invention provides a corresponding detection method by optimizing and adapting the device structure, so that it can achieve trace accurate detection.

Description

Technical Field

[0001] The present invention relates to the field of chemical detection technology, and more particularly to a method and a device for rapidly detecting fatty acid components of trace lipid substances. Background Art

[0002] The detection and identification of trace lipid substances plays a vital role in the study of plant oil metabolism, but the extremely small content of lipid components is often difficult to detect accurately. In the actual operation of detection using conventional methods, it was found that due to the detection system being too large and the sealing being not tight, the internal standard (17:0) would be unstable, which would lead to inaccurate quantification of lipid substances and unstable determination of fatty acid composition. The fatty acid components of trace lipid substances may be detected by lipidome sequencing and nuclear magnetic resonance detection, but the cost is high and the time is long.

[0003] Therefore, how to improve the determination method so that it can meet the requirements of accurate detection of fatty acid components of trace lipid substances is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0004] In view of this, the present invention provides a method and device for rapidly detecting fatty acid components of trace lipid substances, by optimizing the structure of the device and adapting Provides a corresponding detection method to achieve accurate detection of trace amounts .

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A device for rapidly detecting fatty acid components of trace lipid substances comprises a liner tube and a sample bottle, wherein the liner tube is placed in the sample bottle.

[0007] Preferably, the sample bottle is a 1.5 mL GC sample bottle.

[0008] Preferably, the liner tube and the sample bottle are both in plurality.

[0009] The present invention also provides a method for rapidly detecting fatty acid components of trace lipid substances using the device described in the above technical solution, comprising the following steps:

[0010] (1) Add a small amount of oil to be tested into the liner, then add the methyl esterification mixture, and then put the liner into a sample bottle filled with hot water and react in a constant temperature water bath;

[0011] (2) After the reaction is completed, transfer all the solution in the liner tube to another clean empty sample bottle, add NaCl solution and n-hexane solution, vortex centrifuge and extract the supernatant into another clean liner tube;

[0012] (3) The supernatant in the liner is concentrated by nitrogen blowing, and then the liner is placed in a sample bottle and the fatty acid components are detected by gas chromatography.

[0013] Preferably, the methylation mixed solution in step (1) comprises a methylation liquid, an internal standard, an antioxidant BHT and toluene, the methylation liquid is a mixed solution with a volume ratio of methanol:concentrated sulfuric acid=95:5, and the internal standard is heptadecacarbonic acid.

[0014] Furthermore, the volume ratio of methyl esterification liquid: toluene is 10:3, the amount of the internal standard added is 1 μg, and the amount of the antioxidant BHT added is 0.3 μl.

[0015] Preferably, the temperature of the hot water and the constant temperature water bath in step (1) is 85° C., and the water bath time is 2 h.

[0016] Preferably, in step (2), the mass concentration of the NaCl solution is 0.9%, and the volume ratio of methyl esterification liquid: NaCl solution: n-hexane is 2:2:1.

[0017] Preferably, the centrifugal speed in step (2) is 1500 r / min and the centrifugal time is 10 min.

[0018] Preferably, in step (3), the nitrogen blowing is concentrated to 30% of the original volume.

[0019] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method and device for rapidly detecting fatty acid components of trace lipid substances, which has the following beneficial effects:

[0020] The technical solution of the present invention innovatively reduces the size of the methyl esterification reaction system and the reaction container to solve the problems of air tightness and insufficient concentration of the methyl esterification product during the reaction process, and has a simple structure, low cost and easy operation.

[0021] The method of the present invention can be used for the accurate and stable determination of ultra-trace lipid (≤2 μg) fatty acids, and is also applied to the key areas of lipid metabolism research such as the detection of rare fatty acids with extremely low content in plants and lipid structural analysis, providing important assistance for lipid metabolism research. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0023] Figure 1 The device structure and flow chart of the present invention;

[0024] Figure 2 Gas chromatograms of different determination methods;

[0025] Figure 3 Figure 2 shows the fatty acid composition of different determination methods. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] As attached Figure 1 As shown, the device for rapidly detecting fatty acid components of trace lipid substances comprises a liner 1 and a sample bottle 2. The liner 1 is placed in the sample bottle 2 and can be taken out. According to specific experimental detection requirements, there can be multiple sets of liner 1 and sample bottle 2.

[0029] Example 2

[0030] Method for rapidly detecting fatty acid components of trace lipid substances using the device of Example 1:

[0031] Take 2 μg of lipid substances and add them to the liner tube. Add 130 μl of methyl esterification mixture [100 μl of methyl esterification solution (95 vt% methanol, 5 vt% concentrated sulfuric acid), 1 μg of internal standard (mixed with methyl esterification solution), 30 μl of toluene, 0.2 μl of BHT] to the liner tube. Then, insert the liner tube into a sample bottle filled with 85°C hot water and bathe it in a water bath at 85°C for 2 hours.

[0032] Transfer all the solution in the liner to a clean sample bottle, add 100 μl of 0.9% NaCl solution, add 50 μl of n-hexane solution, vortex, centrifuge at 1500 r / min for 10 minutes, and extract the supernatant into a clean liner;

[0033] The supernatant was concentrated to 30% of the original volume using nitrogen gas, and then the liner was plugged into a clean sample vial and the fatty acid composition was determined using a gas chromatograph (GC) with an injection volume of 4 μl.

[0034] FAMEs were analyzed using a capillary column DB-23 (Agilent, 30m). The gas phase analysis parameters were as follows: the capillary column box started at 50°C and maintained for 60s, then heated to 175°C at a heating rate of 35°C / min and maintained for 60s; heated to 230°C at a heating rate of 4°C / min and maintained for 5min; hydrogen flow rate of 40ml / min, air flow rate of 40ml / min, carrier gas split ratio of N2:H2=10:1; injection volume of 2μl.

[0035] The results are attached Figure 2 As shown in C.

[0036] Comparative Example 1

[0037] Take 10 μg of lipid substances and add them to a 1.5 ml sample bottle. Add 260 μl of methyl esterification mixture [200 μl of methyl esterification solution (95 vt% methanol, 5 vt% concentrated sulfuric acid), 2 μg of internal standard (mixed with methyl esterification solution), 60 μl of toluene, 0.4 μl of BHT] to the sample bottle. Then insert the liner into the sample bottle filled with 85°C hot water and bathe in a water bath at 85°C for 2 hours.

[0038] Transfer all the solution in the liner to a clean sample bottle, add 200 μl of 0.9% NaCl solution, add 100 μl of n-hexane solution, vortex, centrifuge at 1500 r / min for 10 minutes, extract the supernatant into a clean liner, and then insert the liner into a clean sample bottle and determine the fatty acid components using a gas chromatograph (GC) with an injection volume of 4 μl.

[0039] The gas chromatography parameters are the same as those in Example 1.

[0040] The results are attached Figure 2 As shown in A.

[0041] Comparative Example 2

[0042] Take 2 μg of lipid substances and add them to a 1.5 ml sample bottle. Add 260 μl of methyl esterification mixture [200 μl of methyl esterification solution (95 vt% methanol, 5 vt% concentrated sulfuric acid), 2 μg of internal standard (mixed with methyl esterification solution), 60 μl of toluene, 0.4 μl of BHT] to the sample bottle. Then insert the liner into the sample bottle filled with 85°C hot water and bathe in a water bath at 85°C for 2 hours.

[0043] Transfer all the solution in the liner to a clean sample bottle, add 200 μl of 0.9% NaCl solution, add 100 μl of n-hexane solution, vortex, centrifuge at 1500 r / min for 10 minutes, extract the supernatant into a clean liner, and then insert the liner into a clean sample bottle and determine the fatty acid components using a gas chromatograph (GC) with an injection volume of 4 μl.

[0044] The gas chromatography was the same as in Example 1.

[0045] The results are attached Figure 2 As shown in B.

[0046] Comparative Example 3

[0047] Take 0.4 μg of lipid substances and add them to the liner tube. Add 130 μl of methyl esterification mixture [100 μl of methyl esterification solution (95 vt% methanol, 5 vt% concentrated sulfuric acid), 1 μg of internal standard (mixed with methyl esterification solution), 30 μl of toluene, 0.2 μl of BHT] to the liner tube. Then, insert the liner tube into a sample bottle filled with 85°C hot water and bathe it in a water bath at 85°C for 2 hours.

[0048] Transfer all the solution in the liner to a clean sample bottle, add 100 μl of 0.9% NaCl solution, add 50 μl of n-hexane solution, vortex, centrifuge at 1500 r / min for 10 minutes, and extract the supernatant into a clean liner;

[0049] The supernatant was concentrated to 30% of the original volume using nitrogen gas, and then the liner was plugged into a clean sample vial and the fatty acid composition was determined using a gas chromatograph (GC) with an injection volume of 4 μl.

[0050] The gas chromatography was the same as in Example 1.

[0051] The results are attached Figure 2 As shown in D.

[0052] Experimental example

[0053] The method of Example 1 was used to determine 2 μg of lipids and 0.4 μg of lipids. The methyl esterification reaction of 2 μg of lipids in a gas phase bottle was used as the control group, and the method of Comparative Example 1 was used as the control group. The results are shown in the attached figure. Figure 3 As shown in the fatty acid composition diagram, it can be seen that the fatty acids determined by this method are close to those of the control group and are closer to the true value.

[0054] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0055] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A device for rapidly detecting fatty acid components of trace lipid substances, characterized in that: The invention comprises a liner tube and a sample bottle, wherein the liner tube is placed in the sample bottle.

2. A device for rapidly detecting fatty acid components of trace lipid substances according to claim 1, characterized in that: The sample bottle is a 1.5 mL GC sample bottle.

3. A device for rapidly detecting fatty acid components of trace lipid substances according to claim 1, characterized in that: The liner tube and the sample bottle are both in plurality.

4. A method for rapid detection of fatty acid components of trace lipid substances, characterized in that: The device according to any one of claims 1 to 3 comprises the following steps: (1) Add a small amount of oil to be tested into the liner, then add the methyl esterification mixture, and then put the liner into a sample bottle filled with hot water and react in a constant temperature water bath; (2) After the reaction is completed, transfer all the solution in the liner tube to another clean empty sample bottle, add NaCl solution and n-hexane solution, vortex centrifuge and extract the supernatant into another clean liner tube; (3) The supernatant in the liner is concentrated by nitrogen blowing, and then the liner is placed in a sample bottle and the fatty acid components are detected by gas chromatography.

5. A method for rapidly detecting fatty acid components of trace lipid substances according to claim 4, characterized in that: The methyl esterification mixed liquid in step (1) comprises a methyl esterification liquid, an internal standard, an antioxidant BHT and toluene. The methyl esterification liquid is a mixed liquid with a volume ratio of methanol:concentrated sulfuric acid=95:5, and the internal standard is heptadecacarbonic acid.

6. A method for rapidly detecting fatty acid components of trace lipid substances according to claim 5, characterized in that: The volume ratio of methyl esterification liquid: toluene is 10:3, the amount of the internal standard added is 1 μg, and the amount of the antioxidant BHT added is 0.3 μl.

7. A method for rapid detection of fatty acid components of trace lipid substances according to claim 4, characterized in that: The temperature of the hot water and the constant temperature water bath in step (1) is 85° C., and the water bath time is 2 h.

8. A method for rapid detection of fatty acid components of trace lipid substances according to claim 4, characterized in that: The mass concentration of the NaCl solution in step (2) is 0.9%, and the volume ratio of methyl esterification liquid: NaCl solution: n-hexane is 2:2:

1.

9. A method for rapidly detecting fatty acid components of trace lipid substances according to claim 4, characterized in that: The centrifugal speed in step (2) is 1500 r / min and the centrifugal time is 10 min.

10. The method for rapid detection of fatty acid components of trace lipid substances according to claim 4, characterized in that: In step (3), nitrogen blowing is used to concentrate the mixture to 30% of the original volume.

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