Method for determining fatty acid content in aquatic products by gas chromatography tandem mass spectrometry

Abstract

The application relates to a method for determining the content of fatty acids in aquatic products by gas chromatography tandem mass spectrometry, comprising the following steps: using n-hexane and dichloromethane as extraction liquid to extract a sample to obtain upper liquid; after the upper liquid is blown dry by nitrogen, isooctane is added for dissolving, and a potassium hydroxide-methanol solution is added for mixing, and then the mixed liquid is clarified in a test tube; then a neutralizing liquid is added, and centrifugal filtration is carried out to obtain supernatant; the supernatant is filtered to obtain a detection liquid for machine testing. According to the technical scheme, the pretreatment method of the aquatic product sample is optimized, the pretreatment time is greatly saved, and the method is simple and fast; meanwhile, the gas chromatography tandem mass spectrometry is used for determination, the quantitative and qualitative results are ensured to be accurate and reliable, and the method has the advantages of saving samples, saving time, accurate and reliable results and the like.

Description

Technical Field

[0001] This invention relates to the field of fatty acid detection technology, specifically to a method for determining the fatty acid content in aquatic products by gas chromatography-tandem mass spectrometry. Background Technology

[0002] Aquatic products are rich in various amino acids and are a healthy food sourced from high-protein, low-fat sources, with most falling into the category of white meat. Compared to red meats like pork, aquatic products such as fish and shrimp have lower fat content, and their fat is higher in unsaturated fatty acids, making them less likely to cause high blood pressure, high cholesterol, and high blood sugar. The fat content of aquatic products is low, with most containing less than 5%, and their unsaturated fatty acid content is far higher than their saturated fat content. Fish contain fatty acids beneficial to human health, such as EPA and DHA, and the content of these essential fatty acids determines the nutritional value of different types of fish. Determining the composition and types of fatty acids in aquatic products, and identifying methods to regulate the content of polyunsaturated fatty acids in aquatic products, are currently hot research topics.

[0003] Long-chain unsaturated fatty acids have attracted widespread attention. The various unsaturated fatty acids contained in aquatic product oils play a vital role in improving the health of microvascular walls and preventing cerebrovascular diseases. However, research on the determination of fatty acid content in aquatic products is relatively limited. Current methods suffer from drawbacks such as complex pretreatment, long processing times, and low accuracy. Therefore, it is necessary to develop a rapid and accurate method for determining the fatty acid content in aquatic products. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for determining the fatty acid content in aquatic products by gas chromatography-tandem mass spectrometry, so as to solve the problems of complex pretreatment, long time consumption and low determination accuracy of existing determination methods.

[0005] This invention is achieved through the following technical solution: A method for determining the fatty acid content in aquatic products by gas chromatography-tandem mass spectrometry, comprising: The sample was extracted using n-hexane and dichloromethane as extraction solvents to obtain the supernatant. After drying the upper layer of liquid with nitrogen, isooctane was added to dissolve it, and potassium hydroxide-methanol solution was added and mixed well. The mixture was then placed in a test tube and clarified. Then add a neutralizing agent, and centrifuge and filter to obtain the supernatant; The supernatant was filtered to obtain the test solution for instrument testing.

[0006] Further specifying the chromatographic conditions to be used: The chromatographic column used was ThermoTG-5MS: 30m*0.25mm*0.25μm; The carrier gas was high-purity helium, and the flow rate was set to 1.1 mL / min under a split ratio of 20:1. Temperature program: Initial column temperature 40℃, hold for 2 min, increase to 200℃ at 30℃ / min, hold for 1 min; increase to 240℃ at 5℃ / min, hold for 2 min; increase to 285℃ at 5℃ / min, hold for 3 min, for a total of 31 min; The injection port temperature is 250℃; The transmission line temperature is 280℃.

[0007] Further specifying the extraction method, the method is as follows: weigh 2-5g of the sample into a 50mL centrifuge tube, add 10-25mL of extraction solution, extract on a water bath shaker for 1-3h, the water bath temperature is 40-50℃, remove and cool, sonicate for 10-20min, and centrifuge at 4500r / min for 5min.

[0008] Further specifying the extraction method, the method is as follows: weigh 2-5g of the sample into a 50mL centrifuge tube, add 10-25mL of extraction solution, homogenize at high speed for 30s, sonicate at 40℃ for 20min, and centrifuge at 4500r / min for 5min.

[0009] Further specified, the ratio of hexane to dichloromethane is 1:1.

[0010] Further specified, the temperature of the nitrogen gas is 40°C.

[0011] Further specified, in the potassium hydroxide-methanol solution, the molar concentration of potassium hydroxide is 2 mol / L; The neutralizing agent is sodium bisulfate, and the neutralization method is as follows: add 1g of sodium bisulfate, vortex mix for 30s, and centrifuge at 4000r / min for 5min.

[0012] Furthermore, the supernatant is filtered using a 0.22 μm filter membrane.

[0013] Further defining the centrifuge tube, the centrifuge tube includes a tube body and a sealing plug for sealing the tube body opening. A support rod is provided on the sealing plug, the upper end of the support rod is connected to the sealing plug, and the lower end extends towards the bottom of the tube body. Multiple annular grinding plates are movably sleeved on the support rod.

[0014] Further specifying, the grinding plate includes a ring made of lightweight material and two grinding discs fixed to the top and bottom surfaces of the ring, the support rod is made of elastic material, and a counterweight is connected to the lower end of the support rod.

[0015] The beneficial effects of this invention are as follows: This invention discloses a method for determining fatty acid content in aquatic products using gas chromatography-tandem mass spectrometry (GC-MS / MS). By optimizing the sample pretreatment method, it significantly reduces pretreatment time and is simple and fast. Furthermore, the use of GC-MS / MS ensures accurate and reliable quantitative and qualitative results, offering advantages such as sample saving, time saving, and accurate and reliable results.

[0016] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0017] Figure 1 The first detection spectrum for shellfish; Figure 2 The second detection spectrum for shellfish; Figure 3 The third detection spectrum for shellfish; Figure 4 The first detection spectrum for shrimp; Figure 5 The second detection spectrum for shrimp; Figure 6 The third detection spectrum for shrimp; Figure 7 The first detection spectrum for fish; Figure 8 The second detection map for fish; Figure 9 The third detection map for fish; Figure 10 This is the front view of the centrifuge tube; Figure 11 for Figure 10 A schematic diagram of the cross-sectional structure of AA; Figure 12 for Figure 11 Enlarged view of B in the middle; Figure 13 This is a schematic diagram of the installation structure of the grinding plate; In the diagram: 1. Tube body; 2. Sealing plug; 3. Support rod; 4. Grinding plate; 5. Ring body; 6. Grinding disc; 7. Counterweight. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0019] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0020] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0021] In the above description of the present invention, it should be noted that the terms "one side," "the other side," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship in which the product of the invention is conventionally placed during use. These terms are used only for the convenience of describing the present invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0022] Furthermore, terms such as "identical" do not imply that components must be absolutely identical; minor differences are permissible. The term "perpendicular" simply means that the positional relationship between components is more perpendicular than "parallel," not that the structure must be perfectly perpendicular; a slight tilt is acceptable.

[0023] Example 1 A method for determining the fatty acid content of aquatic products by gas chromatography-tandem mass spectrometry, comprising the following specific steps: Step 1: Weigh 2-5g of the pulverized sample into a 50mL centrifuge tube, add 10-25mL of extraction solution (prepared by mixing hexane and dichloromethane in a 1:1 ratio), homogenize at high speed for 30s, sonicate at 40℃ for 20min, centrifuge at 4500r / min for 5min to obtain the supernatant. Step 2: Transfer the upper extract to a 50mL centrifuge tube and dry it with nitrogen gas at 40℃; Step 3: Add 5 ml of isooctane (adjust according to fat content) and dissolve thoroughly. Add 0.2 mL of potassium hydroxide-methanol solution (2 mol / L: weigh 13.2 g of potassium hydroxide, dissolve in 80 mL of methanol, cool to room temperature, and make up to 100 mL with methanol). Vortex mix for 1 min and transfer to a test tube to clarify the mixture. Step 4: Add 1g of sodium bisulfate to neutralize the excess potassium hydroxide, vortex mix for 30s, centrifuge at 4000r / min for 5min, and obtain the supernatant; Step 5: Filter the supernatant through a 0.22μm filter membrane and test it on the instrument.

[0024] The chromatographic conditions for gas chromatography-triple quadrupole tandem mass spectrometry analysis are as follows: The ThermoTG-5MS column was selected: 30m*0.25mm*0.25μm; the carrier gas was high-purity helium, and the flow rate was set to 1.1mL / min under the condition of a split ratio of 20:1. Temperature program: Initial column temperature 40℃, hold for 2 min, increase to 200℃ at 30℃ / min, hold for 1 min; increase to 240℃ at 5℃ / min, hold for 2 min; increase to 285℃ at 5℃ / min, hold for 3 min, for a total of 31 min.

[0025] The injection port temperature is 250℃; The transmission line temperature is 280℃.

[0026] Example 2 The difference from Example 1 is as follows: Step 1: Weigh 2-5g of the pulverized sample into a 50mL centrifuge tube, add 10-25mL of extraction solution (prepared by mixing hexane and dichloromethane in a 1:1 ratio), and extract in a water bath at 40-50℃ for 1-3 hours. After cooling, sonicate for 10-20 minutes and centrifuge at 4500r / min for 5 minutes. The centrifuge tube includes a tube body 1 and a sealing plug 2 for sealing the opening of the tube body 1. A support rod 3 is provided on the sealing plug 2. The upper end of the support rod 3 is connected to the sealing plug 2, and the lower end extends towards the bottom of the tube body 1. Multiple annular grinding plates 4 are movably sleeved on the support rod 3.

[0027] The bottom of the tube body 1 is connected to the water bath shaker. The tube body 1 is placed vertically with the tube opening facing upwards. The sealing plug 2 is used to seal the tube opening of the tube body 1. The support rod 3 set on the sealing plug 2 extends upwards into the tube body 1. The multiple grinding plates 4 on it are movably engaged with the support rod 3. In the specific operation, the mud-like sample is mixed with the extract and then suspended in the extract. Multiple grinding plates 4 are movable and cooperate with the support rod 3. During the oscillation, the relative movement between two adjacent grinding plates 4 will occur. The sample suspended in the extract will enter between two adjacent grinding plates 4. The relative movement of the grinding plates 4 will further grind and break it up to improve the extraction effect.

[0028] The grinding plate 6 includes a ring 5 made of lightweight material and two grinding discs 6 fixed to the top and bottom surfaces of the ring 5. The support rod 3 is made of elastic material and a counterweight 7 is connected to the lower end of the support rod 3.

[0029] The support rod 3 is made of elastic materials such as silicone or rubber. Its lower end is equipped with a counterweight 7. During the oscillation, the support rod 3 will be in a state of continuous stretching and elastic contraction and recovery under the drag of the counterweight 7, so as to keep the multiple grinding plates 4 in an active state. Furthermore, the grinding plate 4 consists of a ring body 5 and two grinding discs 6. The ring body 5 is made of lightweight materials such as foam board, and the grinding discs 6 are made of stainless steel sheets with a frosted surface. This reduces the overall structural weight and allows the structure formed by connecting the grinding discs 6 and the ring body 5 to be suspended in the extraction liquid. During the oscillation process, any two adjacent grinding plates 4 can be in a relatively active state.

[0030] The table below shows the mass spectrometry parameters and retention times for each substance:

[0031] Example 2 was used as the detection method for detecting shellfish, shrimp, and fish. The table below shows the test results for the clams;

[0032] The table below shows the test results for the prawns;

[0033] The table below shows the test results for sea bass;

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for determining the fatty acid content in aquatic products by gas chromatography-tandem mass spectrometry, characterized in that: include: The sample was extracted using n-hexane and dichloromethane as extraction solvents to obtain the supernatant. After drying the upper layer of liquid with nitrogen, isooctane was added to dissolve it, and potassium hydroxide-methanol solution was added and mixed well. The mixture was then placed in a test tube and clarified. Then add a neutralizing agent, and centrifuge and filter to obtain the supernatant; The supernatant was filtered to obtain the test solution for instrument testing; The extraction method is as follows: weigh 2-5g of the sample into a 50mL centrifuge tube, add 10-25mL of extraction solution, extract on a water bath shaker for 1-3h, the water bath temperature is 40-50℃, take it out and cool, sonicate for 10-20min, and centrifuge at 4500r / min for 5min. The centrifuge tube includes a tube body and a sealing plug for sealing the tube body opening. A support rod is provided on the sealing plug. The upper end of the support rod is connected to the sealing plug, and the lower end extends towards the bottom of the tube body. Multiple annular grinding plates are movably sleeved on the support rod. The grinding plate includes a ring made of lightweight material and two grinding discs fixed to the top and bottom surfaces of the ring. The support rod is made of elastic material and a counterweight is connected to the lower end of the support rod. During the oscillation process, the support rod will be in a state of continuous stretching and elastic contraction and recovery under the drag of the counterweight, so as to keep multiple grinding plates in an active state; among them, the relative movement of the grinding plates between two adjacent grinding plates further grinds and breaks them down to improve the extraction effect.

2. The method for determining fatty acid content in aquatic products by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The selected chromatographic conditions are: The chromatographic column used was ThermoTG-5MS: 30m*0.25mm*0.25μm; The carrier gas was high-purity helium, and the flow rate was set to 1.1 mL / min under a split ratio of 20:

1. Temperature program: Initial column temperature 40℃, hold for 2 min, increase to 200℃ at 30℃ / min, hold for 1 min; increase to 240℃ at 5℃ / min, hold for 2 min; increase to 285℃ at 5℃ / min, hold for 3 min, for a total of 31 min; The injection port temperature is 250℃; The transmission line temperature is 280℃.

3. The method for determining fatty acid content in aquatic products by gas chromatography-tandem mass spectrometry according to claim 2, characterized in that: The extraction method is as follows: weigh 2-5g of the sample into a 50mL centrifuge tube, add 10-25mL of extraction solution, homogenize at high speed for 30s, sonicate at 40℃ for 20min, and centrifuge at 4500r / min for 5min.

4. The method for determining fatty acid content in aquatic products by gas chromatography-tandem mass spectrometry according to claim 1 or 3, characterized in that: The ratio of n-hexane to dichloromethane is 1:

1.

5. The method for determining fatty acid content in aquatic products by gas chromatography-tandem mass spectrometry according to claim 4, characterized in that: The nitrogen gas temperature is 40°C.

6. The method for determining fatty acid content in aquatic products by gas chromatography-tandem mass spectrometry according to claim 5, characterized in that: In the potassium hydroxide-methanol solution, the molar concentration of potassium hydroxide is 2 mol / L; The neutralizing agent is sodium bisulfate, and the neutralization method is as follows: add 1g of sodium bisulfate, vortex mix for 30s, and centrifuge at 4000r / min for 5min.

7. The method for determining fatty acid content in aquatic products by gas chromatography-tandem mass spectrometry according to claim 6, characterized in that: The supernatant was filtered using a 0.22 μm filter membrane.

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