Method for determining content of amino acid in aquatic product by liquid chromatography-tandem mass spectrometry

Abstract

The invention relates to a method for determining the content of amino acid in an aquatic product by liquid chromatography-tandem mass spectrometry, which comprises the following steps: sample preparation: weighing a sample, putting the sample into a grinder, adding water, grinding, and drying to the initial weight to prepare a detection sample; the method comprises the following steps: weighing a detection sample, putting the detection sample into a digestion tube, adding a hydrochloric acid solution and a phenol reagent, pre-digesting overnight, digesting through a microwave digestion instrument, and removing acid to prepare hydrolysate; adding the hydrolysate into a centrifugal tube, adding ultrapure water, oscillating and shaking uniformly to obtain a uniformly mixed solution; 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 rapid; and meanwhile, the liquid chromatography-tandem mass spectrometry is adopted for determination, so that the accuracy and reliability of quantitative and qualitative results are ensured. The method has the advantages of sample saving, time saving, accurate and reliable result and the like. The method has a certain application value in determination of amino acids in aquatic products, and provides technical support for research on an amino acid metabolism mechanism of aquatic animals.

Description

Technical Field

[0001] The invention relates to the technical field of amino acid detection, and in particular to a method for determining the amino acid content in aquatic products by liquid chromatography tandem mass spectrometry. Background Art

[0002] Aquatic products are rich in a variety of amino acids. They are a high-protein, low-fat healthy food and an important contributor to high-quality protein for humans. They have always been loved by people for their delicious taste and high nutritional value. In nature, there are more than 300 kinds of amino acids, and there are about 20 kinds of amino acids that make up the proteins of organisms. According to whether they can be synthesized in the human body, they are divided into two categories: essential amino acids and non-essential amino acids. Essential amino acids refer to amino acids that cannot be synthesized by the human body (or other vertebrates) or the synthesis speed is far from meeting the needs of the body and must be supplied by food protein. Different animal species require different essential amino acids. For adults, there are 8 essential amino acids, namely: lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, and valine. Aquatic products are rich in essential amino acids. Accurately detecting the composition and content of amino acids in aquatic products is the basis for evaluating the nutritional value of aquatic products and a necessary condition for implementing precise nutrition for the human body.

[0003] At present, the gold standard method for amino acid detection in aquatic products is liquid chromatography in accordance with GB 5009.124-2016 "National Food Safety Standard for the Determination of Amino Acids in Food". This method has the disadvantages of complex pre-treatment, long time consumption, and low determination accuracy. Specifically, the pre-treatment is complicated, and only part of the amino acids in the sample can be extracted by acid hydrolysis once, and amino acids such as tryptophan cannot be detected; second, the derivatization process is complicated, and the requirements for equipment and operation are very high. The derivatization efficiency is affected by the quality of the derivatization reagent; third, the qualitative confirmation of the compound mainly depends on the retention time, which is easily interfered by factors such as the chromatographic column, mobile phase, and matrix, which can easily affect the repeatability of the test results. Summary of the invention

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

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for determining the amino acid content in aquatic products by liquid chromatography tandem mass spectrometry comprises the following steps:

[0007] Sample preparation: weigh the sample, put it into a grinder, grind it with water, and dry it to the initial weight to obtain the test sample;

[0008] Weigh the test sample into a digestion tube, add hydrochloric acid solution and phenol reagent, pre-digest overnight, and then digest and remove the acid in a microwave digestion instrument to obtain a hydrolyzate;

[0009] Add the hydrolyzate into a centrifuge tube, add ultrapure water and shake to obtain a mixed solution;

[0010] Pipette the mixed solution, dilute it with ultrapure water, and shake it with an oscillator to obtain a diluted solution;

[0011] The diluted solution was pipetted into a centrifuge tube, dried with nitrogen, and fully dissolved by adding formic acid solution. The test solution for machine testing was obtained by filtering.

[0012] Put the test liquid on the machine for testing.

[0013] Further defined, liquid chromatography conditions:

[0014] Chromatographic column: T3 (3.0 mm × 100 mm, 1.8 μm);

[0015] Column temperature: 40°C;

[0016] Mobile phase: A is 0.1% formic acid aqueous solution, B is acetonitrile;

[0017] Flow rate: 0.3 mL / min;

[0018] Injection volume: 2.00 μL;

[0019] Mass spectrometry conditions:

[0020] Ion source: electrospray ion source;

[0021] Scanning mode: positive ion mode;

[0022] Detection method: multiple reaction monitoring (MRM);

[0023] Electrospray voltage (IS): 5.5 kV (positive ion mode);

[0024] Ion source temperature (TEM): 550°C;

[0025] Curtain air pressure (CUR): 35.0 psi;

[0026] Collision gas pressure (CAD): 9 psi;

[0027] Spray gas pressure (GS1), auxiliary heating gas pressure (GS2): 55.0 psi.

[0028] It is further specified that the specific preparation method of the hydrolyzate is: weigh 0.1-0.8 g of the test sample into a digestion tube, add 2-4 times the fixed volume of hydrochloric acid solution and phenol reagent of the test sample, pre-digest overnight, digest for 1-5 hours in a microwave digestion apparatus at 150-180°C, take out and cool, and remove the acid in a digestion furnace for 1-5 hours to obtain the result.

[0029] It is further defined that the specific preparation method of the mixed solution is: all the hydrolyzate is transferred to a 50mL centrifuge tube, the volume is made up to 50mL with ultrapure water, and the mixed solution is shaken with an oscillator.

[0030] It is further defined that the specific preparation method of the diluent is: aspirate 1 mL of the mixed solution, dilute it 10 times with ultrapure water, and shake it with an oscillator.

[0031] It is further specified that the specific preparation method of the detector is: absorb 1 ml of the diluent into a 15 mL centrifuge tube, blow dry with nitrogen at 40-50° C., add 1-5 ml of a certain concentration of formic acid solution to fully dissolve, and filter through a 0.22 μm microporous filter membrane.

[0032] It is further defined that the grinder has a first grinding plate and a second grinding plate disposed below the first grinding plate, the center of the second grinding plate is connected to a first transmission shaft, the upper end of the first transmission shaft penetrates the first grinding plate and extends upward, and is rotatably connected to the first grinding plate;

[0033] The top surface of the first grinding plate is connected with a transmission sleeve, the transmission sleeve is coaxially arranged with the first transmission shaft, and a transmission part is arranged between the transmission sleeve and the first transmission shaft;

[0034] The transmission part comprises a driving gear with a single degree of freedom slidingly fitted on the first transmission shaft and a plurality of transmission gears arranged between the intermediate gear and the transmission sleeve, the inner wall of the transmission sleeve is provided with a plurality of transmission ridges extending in the axial direction, and the two sides of the transmission gear are respectively meshed with the driving gear and the transmission ridges;

[0035] A retaining frame is disposed outside the plurality of driven gears, and the driven gears are respectively rotatably matched with the retaining frame through the wheel shaft;

[0036] The upper end of the first transmission shaft is sleeved with a second transmission shaft, the first transmission shaft is slidably fitted in the second transmission shaft with a single degree of freedom, the upper end of the second transmission shaft is rotatably connected to a support plate, and the support plate is mounted with a drive motor that is transmission-connected to the upper end of the second transmission shaft;

[0037] The bottom surface of the support plate is connected with a support sleeve, the retaining frame is fixedly connected to the support sleeve, and the transmission sleeve is movably sleeved outside the support sleeve.

[0038] It is further defined that a protective sleeve is coaxially arranged outside the supporting sleeve, the upper end of the protective sleeve is fixedly connected to the supporting plate, and the upper end of the transmission sleeve is inserted between the supporting sleeve and the protective sleeve;

[0039] A support spring is arranged between the support sleeve and the protective sleeve, and two ends of the support spring are respectively against the upper end of the transmission sleeve and the support plate.

[0040] It is further defined that the outer wall of the first transmission shaft is provided with a plurality of strip-shaped ridges protruding outward and extending axially, and the inner wall of the second transmission shaft is provided with a plurality of strip-shaped grooves extending axially, and the plurality of strip-shaped ridges are respectively located in a plurality of strip-shaped recesses and are slidably fitted.

[0041] It is further defined that the bottom surface of the first grinding plate is provided with a plurality of first bosses extending outward from the center, a first grinding sheet is provided between two adjacent first bosses, and the plurality of first grinding sheets are all protruding from the top surface of the first boss;

[0042] A plurality of first guide holes are formed on the first boss, and the distance between one end of the first guide hole facing the second grinding plate and the center is greater than the distance between the other end and the center;

[0043] The top surface of the second grinding plate is provided with a plurality of second bosses extending outward from the center, a second grinding sheet is provided between two adjacent second bosses, and the plurality of second grinding sheets are all protruding from the top surface of the second boss;

[0044] The second boss is provided with a plurality of second guide holes, and the distance between one end of the second guide hole facing the first grinding plate and the center is greater than the distance between the other end and the center.

[0045] The beneficial effects of the present invention are:

[0046] This method of determining the amino acid content in aquatic products by liquid chromatography-tandem mass spectrometry optimizes the pretreatment method of aquatic product samples, greatly saves pretreatment time, and is simple and quick; at the same time, liquid chromatography-tandem mass spectrometry is used for determination to ensure the accuracy and reliability of quantitative and qualitative results. It has the advantages of saving samples, saving time, and accurate and reliable results. It has certain application value in the determination of amino acids in aquatic products, and provides technical support for the study of amino acid metabolism mechanism of aquatic animals.

[0047] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a front view of the grinder in the present invention;

[0049] Figure 2 for Figure 1 Schematic diagram of the cross-sectional structure of AA;

[0050] Figure 3 is a schematic diagram of the transmission structure of the first grinding plate;

[0051] Figure 4 is a schematic diagram of the transmission structure of the second grinding plate;

[0052] Figure 5 It is a schematic diagram of the transmission structure of the driving gear, the driven gear and the transmission sleeve;

[0053] Figure 6 is a schematic structural diagram of a first grinding plate;

[0054] Figure 7 is a schematic diagram of the matching structure of the first transmission shaft and the second transmission shaft;

[0055] In the figure: 1. shell; 2. support plate; 3. first grinding plate; 4. second grinding plate; 5. first transmission shaft; 6. driving gear; 7. driven gear; 8. transmission sleeve; 9. transmission ridge; 10. retaining frame; 11. second transmission shaft; 12. support sleeve; 13. screen; 14. protective sleeve; 15. support spring; 16. strip ridge; 17. strip groove; 18. first boss; 19. first grinding sheet; 20. first guide hole; 21. second boss; 22. second grinding sheet; 23. second guide hole; 24. driving motor. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0057] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. 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.

[0058] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0059] 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 usually placed when in use, which is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", etc. are only used to distinguish the description, and cannot be understood as indicating or implying relative importance.

[0060] In addition, the term "same" does not mean that the parts must be absolutely the same, but slight differences are allowed. The term "vertical" only means that the positional relationship between the parts is more vertical than "parallel", and does not mean that the structure must be completely vertical, but can be slightly tilted.

[0061] The present invention provides a technical solution: a method for determining the amino acid content in aquatic products by liquid chromatography tandem mass spectrometry, comprising the following steps:

[0062] Sample preparation: weigh the sample, put it into a grinder, grind it with water, and dry it to the initial weight to obtain the test sample;

[0063] Weigh the test sample into a digestion tube, add hydrochloric acid solution and phenol reagent, pre-digest overnight, and then digest and remove the acid in a microwave digestion instrument to obtain a hydrolyzate;

[0064] Add the hydrolyzate into a centrifuge tube, add ultrapure water and shake to obtain a mixed solution;

[0065] Pipette the mixed solution, dilute it with ultrapure water, and shake it with an oscillator to obtain a diluted solution;

[0066] The diluted solution was pipetted into a centrifuge tube, dried with nitrogen, and fully dissolved by adding formic acid solution. The test solution for machine testing was obtained by filtering.

[0067] Put the test liquid on the machine for testing.

[0068] In this embodiment, the liquid chromatography conditions are:

[0069] Chromatographic column: T3 (3.0 mm × 100 mm, 1.8 μm);

[0070] Column temperature: 40°C;

[0071] Mobile phase: A is 0.1% formic acid aqueous solution, B is acetonitrile;

[0072] Flow rate: 0.3 mL / min;

[0073] Injection volume: 2.00 μL;

[0074] Mass spectrometry conditions:

[0075] Ion source: electrospray ion source;

[0076] Scanning mode: positive ion mode;

[0077] Detection method: multiple reaction monitoring (MRM);

[0078] Electrospray voltage (IS): 5.5 kV (positive ion mode);

[0079] Ion source temperature (TEM): 550°C;

[0080] Curtain air pressure (CUR): 35.0 psi;

[0081] Collision gas pressure (CAD): 9 psi;

[0082] Spray gas pressure (GS1), auxiliary heating gas pressure (GS2): 55.0psi;

[0083] In this embodiment, the specific preparation method of the hydrolyzate is as follows: weigh 0.1-0.8 g of the test sample into a digestion tube, add 2-4 times the volume of hydrochloric acid solution and phenol reagent of the test sample, pre-digest overnight, digest for 1-5 hours in a microwave digestion apparatus at 150-180° C., take out and cool, and remove the acid in a digestion furnace for 1-5 hours to obtain the hydrolyzate.

[0084] In this embodiment, the specific preparation method of the mixed solution is: transfer all the hydrolyzate into a 50 mL centrifuge tube, dilute to 50 mL with ultrapure water, and shake with an oscillator.

[0085] In this embodiment, the specific preparation method of the diluent is: take 1 mL of the mixed solution, dilute it 10 times with ultrapure water, and shake it with an oscillator.

[0086] In this embodiment, the specific preparation method of the detector is: absorb 1 ml of the diluent into a 15 mL centrifuge tube, blow dry with nitrogen at 40-50° C., add 1-5 ml of a certain concentration of formic acid solution to fully dissolve, and filter through a 0.22 μm microporous filter membrane.

[0087] Among them, the gradient elution program is shown in the table below;

[0088]

[0089] Mass spectrometry parameters of 21 amino acids:

[0090]

[0091]

[0092] See also Figure 1-7 The grinder comprises a first grinding plate 3 and a second grinding plate 4 disposed below the first grinding plate 3, wherein the center of the second grinding plate 4 is connected to a first transmission shaft 5, the upper end of the first transmission shaft 5 penetrates through the first grinding plate 3 and extends upward, and is rotatably connected to the first grinding plate 3;

[0093] The top surface of the first grinding plate 3 is connected with a transmission sleeve 8, the transmission sleeve 8 is coaxially arranged with the first transmission shaft 5, and a transmission part is arranged between the transmission sleeve 8 and the first transmission shaft 5;

[0094] The transmission part includes a driving gear 6 that is slidably fitted on the first transmission shaft 5 with a single degree of freedom and a plurality of transmission gears disposed between the intermediate gear and the transmission sleeve 8. The inner wall of the transmission sleeve 8 is provided with a plurality of transmission ridges 9 extending in the axial direction. The transmission gears are respectively meshed with the driving gear 6 and the transmission ridges 9 on both sides.

[0095] A retainer 10 is disposed outside the plurality of driven gears 7, and the driven gears 7 are respectively rotatably matched with the retainer 10 through the wheel shaft;

[0096] The upper end of the first transmission shaft 5 is sleeved with a second transmission shaft 11, and the first transmission shaft 5 is slidably fitted in the second transmission shaft 11 with a single degree of freedom. The upper end of the second transmission shaft 11 is rotatably connected to a support plate 2, and a driving motor 24 is installed on the support plate 2 and is transmission-connected to the upper end of the second transmission shaft 11;

[0097] The bottom surface of the support plate 2 is connected with a support sleeve 12 , the retaining frame 10 is fixedly connected to the support sleeve 12 , and the transmission sleeve 8 is movably sleeved outside the support sleeve 12 .

[0098] The second grinding plate 4 has an edge connected with a cylindrical screen 13 extending upward and wrapped around the first grinding plate 3. A space for accommodating a sample is formed between the first grinding plate 3 and the second grinding plate 4. The outer side of the space is closed by the screen 13. At the same time, the housing 1 is connected to the support plate 2, and the support plate 2 serves as a closed structure of the housing 1.

[0099] When in use, the sample is chopped into 0.5cm3 particles, and then placed between the first grinding plate 3 and the second grinding plate 4, and water matching the sample volume is added, the support plate 2 is buckled with the shell 1, and then the drive motor 24 is started. Under the action of the drive motor 24, the first transmission shaft 5 is driven to rotate through the second transmission shaft 11, and then the second grinding plate 4 is rotated; at the same time, the first transmission shaft 5 will drive the driving gear 6 to rotate when rotating, and the transmission gear is connected to rotate through the retaining frame 10, and the retaining frame 10 is fixedly connected to the support sleeve 12, that is, fixedly connected to the support plate 2. When the driving gear 6 rotates, it will drive the driven gear 7 to rotate, and then promote the transmission sleeve 8 and the first grinding plate 3 to rotate, so that the first grinding plate 3 and the second grinding plate 4 are both in a rotating state. Since the rotation of the first grinding plate 3 is driven by the driving gear 6, the driven gear 7 and the transmission sleeve 8, and the second grinding plate 4 is directly driven by the first transmission shaft 5, there is a rotation difference between the rotation speed of the first grinding plate 3 and the rotation speed of the second grinding plate 4, and the sample between the first grinding plate 3 and the second grinding plate 4 can be ground;

[0100] The inner wall of the transmission sleeve 8 forms a transmission structure by arranging transmission ribs 9. The transmission sleeve 8 can slide axially, and can maintain a meshing state with the driven gear 7 in a relatively fixed state during the sliding process through the transmission ribs 9, that is, the first grinding plate 3 squeezes the sample onto the second grinding plate 4 under the action of its own gravity, which can improve the grinding effect of the sample. At the same time, the rotation direction of the first grinding plate 3 and the second grinding plate 4 is the same direction. The same direction rotation can prolong the residence time of the sample between the first grinding plate 3 and the second grinding plate 4, further improving the grinding effect of the sample.

[0101] In this embodiment, a protective sleeve 14 is coaxially arranged outside the support sleeve 12, the upper end of the protective sleeve 14 is fixedly connected to the support plate 2, and the upper end of the transmission sleeve 8 is inserted between the support sleeve 12 and the protective sleeve 14;

[0102] A support spring 15 is disposed between the support sleeve 12 and the protective sleeve 14 , and two ends of the support spring 15 are respectively against the upper end of the transmission sleeve 8 and the support plate 2 .

[0103] At the same time, a support spring 15 is arranged between the support sleeve 12 and the support plate 2. Under the action of the support spring 15, a downward thrust is applied to the support sleeve 12, that is, a downward thrust is applied to the first grinding plate 3, so that the first grinding plate 3 and the second grinding plate 4 are kept in a squeezed and fitted state, and the pressure applied to the sample is increased together with the self-gravity of the first grinding plate 3, thereby further improving the grinding effect of the sample.

[0104] In this embodiment, the outer wall of the first transmission shaft 5 is provided with a plurality of strip-shaped ridges 16 protruding outward and extending axially, and the inner wall of the second transmission shaft 11 is provided with a plurality of strip-shaped grooves 17 extending axially, and the plurality of strip-shaped ridges 16 are respectively located in a plurality of strip-shaped recesses and are slidably fitted.

[0105] The strip convex ribs 16 cooperate with the strip grooves 17. When the first transmission shaft 5 and the second transmission shaft 11 move relative to each other in the axial direction, the strip convex ribs 16 can slide along the strip grooves 17. When the second transmission shaft 11 drives the first transmission shaft 5 to rotate, the strip convex ribs 16 and the strip grooves 17 will form a rotation limiting structure, so that the first transmission shaft 5 can rotate synchronously with the second transmission shaft 11.

[0106] Similarly, the matching manner of the driving gear 6 and the first transmission shaft 5 can be the same as this manner.

[0107] In this embodiment, the bottom surface of the first grinding plate 3 is provided with a plurality of first bosses 18 extending outward from the center, and a first grinding sheet 19 is provided between two adjacent first bosses 18, and the plurality of first grinding sheets 19 are all protruding from the top surface of the first boss 18;

[0108] The first boss 18 is provided with a plurality of first guide holes 20, wherein the distance between one end of the first guide hole 20 facing the second grinding plate 4 and the center is greater than the distance between the other end and the center;

[0109] The top surface of the second grinding plate 4 is provided with a plurality of second bosses 21 extending outward from the center, and a second grinding sheet 22 is provided between two adjacent second bosses 21, and the plurality of second grinding sheets 22 are all protruding from the top surface of the second boss 21;

[0110] A plurality of second guide holes 23 are formed on the second boss 21 . The distance between the center of the second guide hole 23 and one end facing the first grinding plate 3 is greater than the distance between the center of the other end.

[0111] An installation space for installing the first grinding sheet 19 is formed between the two first bosses 18, and the first grinding sheet 19 protrudes from the bottom surface of the first boss 18, so that the corresponding position of the first boss 18 forms a groove structure on the planes of the first grinding sheets 19. During the sample grinding process, the added water and sample mixture will enter the groove structure, and the first guide hole 20 will form a flow channel. Under the action of the centrifugal force when the first grinding sheet 19 rotates, the first guide hole 20 will introduce water between the first grinding sheet 19 and the second grinding sheet 22, so as to replenish water for the sample between the first grinding sheet 19 and the second grinding sheet 22. At the same time, the sample flowing out of the first grinding sheet 19 and the second grinding sheet 22 will be sent back to the first grinding sheet 19 and the second grinding sheet 22 for grinding, so as to further improve the grinding effect of the sample;

[0112] Similarly, the second guide hole 23 constitutes another flow channel. Under the action of the centrifugal force when the second grinding plate 22 rotates, the second guide hole 23 guides the water into the space between the first grinding plate 19 and the second grinding plate 22, so as to replenish the water of the sample between the first grinding plate 19 and the second grinding plate 22. At the same time, the sample flowing out of the space between the first grinding plate 19 and the second grinding plate 22 is sent back into the space between the first grinding plate 19 and the second grinding plate 22 for grinding.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for determining the amino acid content in aquatic products by liquid chromatography-tandem mass spectrometry. Features: The steps include: Sample preparation: weigh the sample, put it into a grinder, grind it with water, and dry it to the initial weight to obtain the test sample; Weigh the test sample into a digestion tube, add hydrochloric acid solution and phenol reagent, pre-digest overnight, and then digest and remove the acid in a microwave digestion instrument to obtain a hydrolyzate; Add the hydrolyzate into a centrifuge tube, add ultrapure water and shake to obtain a mixed solution; Pipette the mixed solution, dilute it with ultrapure water, and shake it with an oscillator to obtain a diluted solution; The diluted solution was pipetted into a centrifuge tube, dried with nitrogen, and fully dissolved by adding formic acid solution. The test solution for machine testing was obtained by filtering. Put the test liquid on the machine for testing.

2. The method for determining the amino acid content in aquatic products by liquid chromatography tandem mass spectrometry according to claim 1, Features: Liquid chromatography conditions: Chromatographic column: T3 (3.0 mm × 100 mm, 1.8 μm); Column temperature: 40°C; Mobile phase: A is 0.1% formic acid aqueous solution, B is acetonitrile; Flow rate: 0.3 mL / min; Injection volume: 2.00 μL; Mass spectrometry conditions: Ion source: electrospray ion source; Scanning mode: positive ion mode; Detection method: multiple reaction monitoring (MRM); Electrospray voltage (IS): 5.5 kV (positive ion mode); Ion source temperature (TEM): 550°C; Curtain air pressure (CUR): 35.0psi; Collision gas pressure (CAD): 9 psi; Spray gas pressure (GS1), auxiliary heating gas pressure (GS2): 55.0psi.

3. The method for determining the amino acid content in aquatic products by liquid chromatography tandem mass spectrometry according to claim 1, Features: The specific preparation method of the hydrolyzate is as follows: weigh 0.1-0.8g of the test sample into a digestion tube, add 2-4 times the volume of hydrochloric acid solution and phenol reagent of the test sample, pre-digest overnight, digest for 1-5h in a microwave digestion apparatus at 150-180°C, take out and cool, and remove the acid in a digestion furnace for 1-5h to obtain the hydrolyzate.

4. The method for determining the amino acid content in aquatic products by liquid chromatography tandem mass spectrometry according to claim 1, Features: The specific preparation method of the mixed solution is: transfer all the hydrolyzate into a 50 mL centrifuge tube, make up to 50 mL mark with ultrapure water, and shake with an oscillator to obtain the mixed solution.

5. The method for determining the amino acid content in aquatic products by liquid chromatography tandem mass spectrometry according to claim 1, Features: The specific preparation method of the diluent is: take 1 mL of the mixed solution, dilute it 10 times with ultrapure water, and shake it with an oscillator.

6. The method for determining the amino acid content in aquatic products by liquid chromatography tandem mass spectrometry according to claim 1, Features: The specific preparation method of the detector is as follows: 1 ml of the diluent is taken into a 15 mL centrifuge tube, dried with nitrogen at 40-50° C., 1-5 ml of a certain concentration of formic acid solution is added to fully dissolve, and filtered through a 0.22 μm microporous filter membrane.

7. The method for determining the amino acid content in aquatic products by liquid chromatography tandem mass spectrometry according to any one of claims 1 to 6, Features: The grinder comprises a first grinding plate and a second grinding plate disposed below the first grinding plate, wherein the center of the second grinding plate is connected to a first transmission shaft, an upper end of the first transmission shaft penetrates through the first grinding plate and extends upward, and is rotatably connected to the first grinding plate; The top surface of the first grinding plate is connected with a transmission sleeve, the transmission sleeve is coaxially arranged with the first transmission shaft, and a transmission part is arranged between the transmission sleeve and the first transmission shaft; The transmission part comprises a driving gear with a single degree of freedom slidingly fitted on the first transmission shaft and a plurality of transmission gears arranged between the intermediate gear and the transmission sleeve, the inner wall of the transmission sleeve is provided with a plurality of transmission ridges extending in the axial direction, and the two sides of the transmission gear are respectively meshed with the driving gear and the transmission ridges; A retaining frame is disposed outside the plurality of driven gears, and the driven gears are respectively rotatably matched with the retaining frame through the wheel shaft; The upper end of the first transmission shaft is sleeved with a second transmission shaft, the first transmission shaft is slidably fitted in the second transmission shaft with a single degree of freedom, the upper end of the second transmission shaft is rotatably connected to a support plate, and the support plate is mounted with a drive motor that is transmission-connected to the upper end of the second transmission shaft; The bottom surface of the support plate is connected with a support sleeve, the retaining frame is fixedly connected to the support sleeve, and the transmission sleeve is movably sleeved outside the support sleeve.

8. The method for determining the amino acid content in aquatic products by liquid chromatography tandem mass spectrometry according to claim 7, Features: A protective sleeve is coaxially arranged outside the supporting sleeve, the upper end of the protective sleeve is fixedly connected to the supporting plate, and the upper end of the transmission sleeve is inserted between the supporting sleeve and the protective sleeve; A support spring is arranged between the support sleeve and the protective sleeve, and two ends of the support spring are respectively against the upper end of the transmission sleeve and the support plate.

9. The method for determining the amino acid content in aquatic products by liquid chromatography tandem mass spectrometry according to claim 7, Features: The outer wall of the first transmission shaft is provided with a plurality of strip-shaped ridges protruding outward and extending axially, and the inner wall of the second transmission shaft is provided with a plurality of strip-shaped grooves extending axially. The plurality of strip-shaped ridges are respectively located in the plurality of strip-shaped grooves and are slidably fitted.

10. The method for determining the amino acid content in aquatic products by liquid chromatography tandem mass spectrometry according to claims 7 to 9, Features: The bottom surface of the first grinding plate is provided with a plurality of first bosses extending outward from the center, a first grinding sheet is provided between two adjacent first bosses, and the plurality of first grinding sheets are all protruding from the top surface of the first boss; A plurality of first guide holes are formed on the first boss, and the distance between one end of the first guide hole facing the second grinding plate and the center is greater than the distance between the other end and the center; The top surface of the second grinding plate is provided with a plurality of second bosses extending outward from the center, a second grinding sheet is provided between two adjacent second bosses, and the plurality of second grinding sheets are all protruding from the top surface of the second boss; The second boss is provided with a plurality of second guide holes, and the distance between one end of the second guide hole facing the first grinding plate and the center is greater than the distance between the other end and the center.

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