Method and device for detecting instant branched chain amino acid free monomer

Abstract

The invention discloses a method for detecting an instant branched chain amino acid free monomer. The method comprises the following steps: preparing water, acetonitrile, methanol, a hydrochloric acid solution, an acetic acid solution, a sodium hydroxide solution, an OPA solution, a borate buffer solution, an acetate buffer solution I, an acetate buffer solution II, a leucine reference substance, an isoleucine reference substance and a valine reference substance; the device comprises a high performance liquid chromatograph, a liquid chromatography processor, a chromatographic column, an analytical balance and a centrifugal tube, the detection method comprises the following steps in sequence: preparing a contrast solution, preparing a sample solution, determining the sample solution and the contrast solution, and calculating a result. And an efficient quantitative detection scheme of the instant branched chain amino acid free monomer is provided.

Description

Technical Field

[0001] The present invention belongs to the field of amino acid detection. Background Art

[0002] The content of free monomers in instant branched-chain amino acid products is one of the important indicators to measure their quality; by detecting the content of free monomers, the purity, stability and effectiveness of the products can be evaluated to ensure that the products meet relevant standards and regulations; understanding the content of free monomers in instant branched-chain amino acid products helps to formulate personalized nutritional supplementation plans; for people who need to supplement branched-chain amino acids, the intake can be adjusted according to the test results to meet the nutritional needs of the body; certain chronic diseases or metabolic disorders may affect the metabolism of branched-chain amino acids; by regularly detecting the content of free monomers of instant branched-chain amino acids, these potential health problems can be detected in time, winning time for early intervention and treatment; in the fields of biomedical research, nutrition, ecology, etc., the detection of free amino acids is of great significance for understanding the state of life activities, exploring the mechanisms of disease occurrence, guiding nutritional intake, etc. The detection of free monomers of instant branched-chain amino acids also helps to promote scientific research and application in these fields;

[0003] This solution provides an efficient quantitative detection solution for free monomers of instant branched-chain amino acids. Summary of the Invention

[0004] Object of the Invention: In order to overcome the deficiencies in the prior art, the present invention provides a detection method and device for free monomers of instant branched-chain amino acids, providing an efficient quantitative detection solution for free monomers of instant branched-chain amino acids.

[0005] Technical Solution: To achieve the above object, the detection method for free monomers of instant branched-chain amino acids of the present invention prepares water, acetonitrile, methanol, hydrochloric acid solution, acetic acid solution, sodium hydroxide solution, OPA solution, borate buffer solution, acetate buffer solution I, acetate buffer solution II, leucine reference substance, isoleucine reference substance, valine reference substance; high performance liquid chromatograph, liquid chromatography processor, chromatographic column, analytical balance and centrifuge tube; the detection steps are successively "preparing a control solution", "preparing a sample solution", "determining the sample solution and the control solution", and "result calculation".

[0006] Furthermore, the method of preparing the OPA solution immediately before use: Weigh 0.08 g of phthalaldehyde, accurate to 0.0001 g, add 7 mL of boric acid buffer solution and 1 mL of acetonitrile to dissolve, add 125 μL of 3-mercaptopropionic acid, and mix well.

[0007] Furthermore, the preparation method of the borate buffer solution: Weigh 24.7 g of boric acid, dissolve it in 800 mL of water, adjust the pH to 10.2 with sodium hydroxide solution, and dilute it with water to 1000 mL; The preparation method of the acetate buffer solution I: Weigh 6.0 g of sodium acetate, dissolve it in 4000 mL of water, add 800 μL of triethylamine and 24 mL of tetrahydrofuran, mix well, and adjust the pH to 7.2 with acetic acid solution; The preparation method of the acetate buffer solution II: Weigh 10.9 g of sodium acetate, dissolve it in 800 mL of water, and adjust the pH to 7.2 with acetic acid solution.

[0008] Furthermore, the method for "preparing the reference solution": Weigh 0.1 g of the reference substance, accurate to 0.0001 g, dissolve it in water and make the volume up to 100 mL respectively; The method for "preparing the sample solution": Weigh 0.5 g of the sample, accurate to 0.0001 g, dissolve it in water and make the volume up to 250 mL respectively.

[0009] Furthermore, the method for "determining the sample solution and the reference solution":

[0010] Determination of the sample solution

[0011] Precisely pipette 10 μL of the sample solution into centrifuge tube 3, precisely add 50 μL of the borate buffer solution, precisely add 10 μL of the OPA solution, and add 330 μL of water to obtain "the mixed liquid to be determined 5". The primary amino acids in "the mixed liquid to be determined 5" can react with o-phthalaldehyde (OPA) to form OPA-amino acids. Then, after mixing the mixed liquid to be determined 5 evenly, immediately precisely measure 40 μL and inject it into the liquid chromatograph, and record the chromatogram.

[0012] Determination of the reference solution

[0013] Precisely measure 10 μL of the reference solution into a centrifuge tube, precisely add 50 μL of the borate buffer solution, precisely add 10 μL of the OPA solution, and add 330 μL of water. Similarly, obtain "the mixed liquid to be determined", mix well, immediately precisely measure 40 μL and inject it into the liquid chromatograph, and record the chromatogram.

[0014] Furthermore, "result calculation":

[0015] The contents of L-leucine, L-isoleucine, and L-valine are expressed as ω i in percentage (%), and are calculated according to the following formula:

[0016]

[0017] In the formula:

[0018] Ai—the peak areas of L-leucine, L-isoleucine, and L-valine in the sample;

[0019] cs——Concentration of the control solution of L-leucine, L-isoleucine, and L-valine, in nanomoles per milliliter (nmol / mL);

[0020] V——The volume for volume-fixing of the sample solution, in milliliters (mL);

[0021] M i ——Molar mass of L-leucine, L-isoleucine, and L-valine, in grams per mole (g / mol) (M(L-leucine,

[0022] L-isoleucine) = 131.17, M(L-valine) = 117.15);

[0023] f——Dilution factor;

[0024] As——Peak area of the control solution of L-leucine, L-isoleucine, and L-valine;

[0025] m——Numerical value of the sample mass, in grams (g);

[0026] 10 9 ——Conversion factor.

[0027] The test results are expressed as the arithmetic mean of parallel determination results, and the results are retained to three significant figures.

[0028] Total amino acid content: The total amino acid content is the sum of the contents of each monomer amino acid;

[0029] Precision: The absolute difference between two independent determination results obtained under repeatability conditions should not be greater than 3% of the arithmetic mean.

[0030] Furthermore, the wall of the conical shell head at the lower end of the centrifuge tube is evenly perforated with liquid seepage holes, and a conical elastic silica gel film is attached to the inner wall surface of the conical shell head. The upper film contour of the conical elastic silica gel film is integrally connected to the upper inner wall edge contour of the conical shell head; a number of paddle pieces are arranged in a circumferential array at the lower inner wall of the cylindrical part of the centrifuge tube; it also includes a rotating cup that cooperates with the improved centrifuge tube. The lower end of the rotating cup is coaxially fixedly connected to the vertical output shaft of the motor with forward and reverse functions; pre-installed water at 32°C to 38°C is pre-injected into the cup cavity inside the rotating cup. A piston seal ring is arranged along the upper inner wall edge of the rotating cup, and the inner diameter of the piston seal ring is adapted to the outer diameter of the cylindrical part of the centrifuge tube; when the conical shell head at the lower end of the centrifuge tube is just completely coaxially inserted downward into the rotating cup, the liquid level of the pre-installed water rises to the upper position of the rotating cup, and the piston seal ring is hermetically sleeved on the lower outer wall of the cylindrical part of the centrifuge tube, and a static friction force is formed between the piston seal ring and the centrifuge tube.

[0031] Further, during the "determination of the sample solution", after gradually and quantitatively adding the sample solution, borate buffer solution, OPA solution, and water into the centrifuge tube, the "mixed liquid to be determined" obtained accumulates inside the conical elastic silica gel film at the bottom end of the centrifuge tube. Subsequently, immediately use the hand or a tool to forcefully push down the centrifuge tube, causing the centrifuge tube to displace downward until the lower end of the centrifuge tube is in limited contact with the bottom of the rotating cup; after covering the centrifuge tube cap, immediately control the output shaft of the motor to perform periodic alternating forward and reverse rotations, and the time for the periodic alternating forward and reverse rotations of the output shaft of the motor is controlled within 5 s; after the reaction ends, immediately forcefully pull up the centrifuge tube after opening the centrifuge tube cap, causing the centrifuge tube to displace upward relative to the rotating cup to the initial position, and immediately quantitatively inject the reacted "mixed liquid to be determined" into the liquid chromatograph using a pipette.

[0032] Further, during the "determination of the sample solution", 30 μL of tris(2-carboxy)phosphine (TCEP) solution is further added to the "mixed liquid to be determined" obtained by gradually and quantitatively adding the sample solution, borate buffer solution, OPA solution, and water into the centrifuge tube.

[0033] Beneficial effects: The present invention provides an efficient quantitative detection scheme for instant branched-chain amino acid free monomers. At the same time, during the "determination of the sample solution", the mixing and reaction of the mixed liquid to be determined can be quickly completed within a short time, avoiding problems such as inaccurate detection caused by insufficient reaction (insufficient mixing) and too long reaction time. Description of the Drawings

[0034] Figure 1 The main reaction formula during the detection process of this scheme;

[0035] Figure 2 It is a flowchart of the detection method;

[0036] Figure 3 It is a schematic structural diagram of the newly designed centrifuge tube and rotating cup;

[0037] Figure 4 It is a schematic structural diagram of two states of the newly designed centrifuge tube and rotating cup. Detailed Embodiments

[0038] The present invention will be further described in detail below with reference to the accompanying drawings.

[0039] In the presence of a thiol reagent, a primary amino acid can react with o-phthalaldehyde (OPA) to form an OPA-amino acid. After separation by reverse high-performance liquid chromatography, it is detected with an ultraviolet detector at a wavelength of 338 nm. Within a certain concentration range (25 pmol - 2500 pmol), its absorbance is proportional to the amino acid concentration. Quantitative analysis is performed by external standard method using an ultraviolet detector. In an environment of 3-mercaptopropionic acid, the simplified reaction formula for the reaction of an amino acid (represented by the general formula RCH(NH2)COOH) with OPA to form an OPA-amino acid derivative can be expressed as shown in Figure 1 As shown, in the above reaction formula, R represents the side chain group of the amino acid; actually, the product is an isoindole derivative, which is simplified to be represented as "OPA-RCH(NH-CO-CH2-S-CH2CH2COOH)" for simplicity.

[0040] Based on the above principle, the following detection scheme is designed in this case, and the flow chart is as shown in Figure 2 As follows:

[0041] Reagents and materials:

[0042] Water: GB / T 6682, grade I water.

[0043] Acetonitrile: chromatographically pure.

[0044] Methanol: chromatographically pure.

[0045] Hydrochloric acid solution: 0.1 mol / L.

[0046] Acetic acid solution: Weigh 2.0 g of acetic acid and dissolve it in 98 mL of water.

[0047] Sodium hydroxide solution: Weigh 40.0 g of sodium hydroxide and dissolve it in 60 mL of water.

[0048] OPA solution: Weigh 0.08 g of o-phthalaldehyde, accurate to 0.0001 g, dissolve it in 7 mL of boric acid buffer solution and 1 mL of acetonitrile, add 125 μL of 3-mercaptopropionic acid, mix well, and prepare it freshly before use.

[0049] Borate buffer solution: Weigh 24.7 g of boric acid, dissolve it in 800 mL of water, adjust the pH to 10.2 with sodium hydroxide solution, and dilute it to 1000 mL with water.

[0050] Acetate buffer solution I: Weigh 6.0 g of sodium acetate, dissolve it in 4000 mL of water, add 800 μL of triethylamine and 24 mL of tetrahydrofuran, mix well, and adjust the pH to 7.2 with acetic acid solution.

[0051] Acetate buffer solution II: Weigh 10.9 g of sodium acetate, dissolve it in 800 mL of water, and adjust the pH to 7.2 with acetic acid solution.

[0052] Leucine reference substance (C6H 13 NO2, CAS No.: 61-90-5): Purity ≥ 99.0%, or a reference substance certified by the state and granted a certificate of reference material.

[0053] Isoleucine reference substance (C6H 13 NO2, CAS No.: 73-32-5): Purity ≥ 99.0%, or a reference substance certified by the state and granted a certificate of reference material.

[0054] Valine reference substance (C5H 11 NO2, CAS No.: 72-18-4): Purity ≥ 99.0%, or a reference substance certified by the state and granted a certificate of reference material.

[0055] Instruments and equipment:

[0056] High performance liquid chromatograph: Equipped with an ultraviolet detector and an auto-sampler.

[0057] Liquid chromatography processor: Chemical workstation.

[0058] Chromatographic column: C18 (packed with octadecylsilyl-bonded silica gel), 250 mm × 4.6 mm, 5 μm, or equivalent performance.

[0059] Analytical balance: Sensitivity of 0.0001 g.

[0060] Centrifuge tube: 1.5 mL.

[0061] Procedures:

[0062] Preparation of reference solution: Weigh 0.1 g of the reference substance, accurate to 0.0001 g, dissolve it in water and make the volume up to 100 mL respectively.

[0063] Preparation of test solution: Weigh 0.5 g of the test sample, accurate to 0.0001 g, dissolve it in water and make the volume up to 250 mL respectively.

[0064] Instrument reference conditions:

[0065] Detection wavelength: 338 nm.

[0066] Temperature: 35 °C.

[0067] Mobile phase A: Acetate buffer solution I.

[0068] Mobile phase B: Acetate buffer solution II + acetonitrile + methanol = 800 + 1400 + 1800.

[0069] Mobile phase gradient elution program is shown in Table A.1.

[0070] Table A.1 Gradient elution program

[0071] Time / min Mobile phase A / % Mobile phase B / % Flow rate / (mL / min) 0.0 100 0 1.0 17.0 70 30 1.0 35.0 22 78 1.0 35.1 0 100 1.2 46.0 0 100 1.2 46.1 100 0 1.0 49.1 100 0 1.0

[0072] Determination of sample solution

[0073] Precisely pipette 10 μL of the sample solution and place it in centrifuge tube 3. Precisely add 50 μL of borate buffer solution, precisely add 10 μL of OPA solution, and add 330 μL of water to obtain "mixed liquid 5 to be determined". The primary amino acids in "mixed liquid 5 to be determined" can react with o-phthalaldehyde (OPA) to form OPA-amino acids. Then, after mixing "mixed liquid 5 to be determined", immediately precisely measure 40 μL and inject it into the liquid chromatograph to record the chromatogram.

[0074] Determination of control solution

[0075] Precisely measure 10 μL of the reference solution and place it in centrifuge tube 3. Precisely add 50 μL of borate buffer solution, precisely add 10 μL of OPA solution, and add 330 μL of water to also obtain "mixed liquid 5 to be determined". Mix well and immediately precisely measure 40 μL and inject it into the liquid chromatograph to record the chromatogram.

[0076] Note: OPA-amino acids are unstable, and sample injection analysis should be carried out immediately after the derivatization reaction ends.

[0077] During the "determination of sample solution" process, the OPA-amino acids obtained from the reaction of the mixed liquid 5 to be determined in centrifuge tube 3 are unstable. The mixing and reaction of the mixed liquid 5 to be determined need to be completed quickly within a short time. Whether the reaction is insufficient (inadequate mixing) or the reaction time is too long, it will cause problems with inaccurate detection. Therefore, a more efficient and rapid mixing reaction needs to be implemented for the mixed liquid 5 to be determined in centrifuge tube 3;

[0078] As Figure 3 shown, the lower end of the common centrifuge tube 3 is a conical shell head 6 structure. During the above "determination of sample solution" process, the total amount of the mixed liquid 5 to be mixed added into centrifuge tube 3 is only 400 μL. The final mixed liquid 5 to be determined added into centrifuge tube 3 will gather in the conical shell head 6. Due to the small liquid volume, it is not conducive to manual or machine mixing. If the method of alternately rotating centrifuge tube 3 forward and backward is adopted, since the mixed liquid 5 to be determined gathers near the axis of centrifuge tube 3, the rotational linear velocity of the mixed liquid 5 to be determined is low. It is very difficult to fully mix the mixed liquid 5 within a very short time by alternately rotating centrifuge tube 3 forward and backward and using the inertia of the liquid itself and the friction with the inner wall. To enable the mixed liquid 5 to be determined in centrifuge tube 3 to be efficiently mixed and reacted in a shorter time, the following improvements are made to centrifuge tube 3:

[0079] On the wall of the conical shell head 6 of the new design in this case, liquid seepage holes 7 are evenly hollowed out. An elastic silicone film 8 in the shape of a conical shell is attached to the inner wall surface of the conical shell head 6. The film thickness of the elastic silicone film 8 in the shape of a conical shell is 0.5 mm. The upper film contour 4 of the elastic silicone film 8 in the shape of a conical shell is integrally heat-pressed and sealed with the inner wall of the upper end of the conical shell head 6 along the contour. On the lower inner wall of the cylindrical part 3a of the centrifuge tube 3, a number of paddle pieces 2 are arranged in a circumferential and integral manner. It also includes a rotating cup 10 that cooperates with the improved centrifuge tube 3. The lower end of the rotating cup 10 is coaxially and fixedly connected to a vertical motor output shaft 11 with a forward and reverse function. Pre-installed water 9 at 32 °C to 38 °C is pre-injected into the cup cavity inside the rotating cup 10. Along the wheel on the upper inner wall of the rotating cup 10, a piston seal ring 13 made of silicone material is provided. The inner diameter of the piston seal ring 13 is adapted to the outer diameter of the cylindrical part 3a of the centrifuge tube 3. When the conical shell head 6 at the lower end of the centrifuge tube 3 is just completely coaxially inserted downward into the rotating cup 10, it is the initial state, as Figure 4 shown on the left. The liquid level of the pre-installed water 9 rises to the upper end position of the rotating cup 10, and the piston seal ring 13 is hermetically sleeved on the lower outer wall of the cylindrical part 3a of the centrifuge tube 3. A static frictional force is formed between the piston seal ring 13 and the centrifuge tube 3, and its maximum static frictional force can be adjusted through material characteristics and the tightness of the sleeve during the design stage.

[0080] The high-efficiency and rapid mixing reaction principle and working process of the newly designed centrifuge tube 3:

[0081] In the initial state, the conical shell head 6 at the lower end of the centrifuge tube 3 is just completely coaxially inserted downward into the rotating cup 10, as Figure 4 shown in the left figure. The liquid level of the pre-installed water 9 is at the upper end of the rotating cup 10, and the piston seal ring 13 is hermetically sleeved on the lower outer wall of the cylindrical part 3a of the centrifuge tube 3. During the "determination of the sample solution", after gradually and quantitatively adding the sample solution, borate buffer solution, OPA solution and water into the centrifuge tube 3, the "mixed liquid to be determined 5" is gathered inside the elastic silicone film 8 in the shape of a conical shell at the bottom end of the centrifuge tube 3. Subsequently, immediately push down the centrifuge tube 3 forcefully by hand or tool, so that the centrifuge tube 3 moves downward until the lower end of the centrifuge tube 3 is in limit contact with the bottom of the cup inside the rotating cup 10. During the process of the centrifuge tube 3 moving downward, the pre-installed water 9 inside the rotating cup 10 is squeezed, so that the pre-installed water 9 originally existing in the rotating cup 10 is squeezed through the liquid seepage holes 7 evenly hollowed out on the wall of the conical shell head 6 and into the space between the inner wall of the conical shell head 6 and the elastic silicone film 8 in the shape of a conical shell, so that the elastic silicone film 8 in the shape of a conical shell deforms upward under the extrusion of the pre-installed water 9 until the elastic silicone film 8 in the shape of a conical shell with the original tip facing downward deforms upward into a shape with the tip facing upward, as Figure 4As shown in the right figure, at this time, the "mixed liquid to be measured 5" is gathered in the annular water channel 5a between the conical elastic silica gel film 8 and the cylindrical part 3a of the centrifuge tube 3. The "mixed liquid to be measured 5" in the annular water channel 5a is farther from the axis of the centrifuge tube 3 than in the initial state, thus bringing a greater rotational linear velocity for subsequent rotation. At the same time, the static friction force between the piston seal ring 13 and the centrifuge tube 3 at this time keeps the centrifuge tube 3 and the rotating cup 10 still in a synchronous state; as Figure 4 the right figure of

[0082] After the above process is completed, cover the centrifuge tube cap 14, and immediately control the output shaft 11 of the motor to perform periodic alternating forward and reverse rotations, so that the centrifuge tube 3 and the rotating cup 10 synchronously perform periodic alternating forward and reverse rotations. The "mixed liquid to be measured 5" in the annular water channel 5a reciprocates, surges and tears back and forth in the circumferential direction under its own inertia and the frictional shear and agitation of the inner wall of the alternately rotating annular water channel 5a and several blades 2, so that the primary amino acids in the "mixed liquid to be measured 5" and o-phthalaldehyde OPA react quickly and fully in a short time under the environment of tumbling, surging and tearing, generating OPA-amino acids; the time for the output shaft 11 of the motor to perform periodic alternating forward and reverse rotations is controlled within 5 s;

[0083] After the reaction is completed, it is necessary to immediately quantitatively inject the reacted "mixed liquid to be measured 5" into the liquid chromatograph by a pipette. However, the "mixed liquid to be measured 5" gathered in the annular water channel 5a at this time is not convenient for the pipette to pick up; therefore, after opening the centrifuge tube cap 14, immediately pull the centrifuge tube 3 upward forcefully, so that the centrifuge tube 3 is displaced upward relative to the rotating cup 10 to the initial position. During the process of the centrifuge tube 3 being displaced upward relative to the rotating cup 10, a negative pressure is generated on the lower side of the conical elastic silica gel film 8, and the conical elastic silica gel film 8 re-adheres to the inner wall surface of the conical head 6 under the action of the negative pressure, as Figure 4 the left figure of Figure 4 At this time, the completely reacted "mixed liquid to be measured 5" re-aggregates inside the conical elastic silica gel film 8 at the bottom end of the centrifuge tube 3, as

[0084] Result calculation

[0085] The contents of L-leucine, L-isoleucine, and L-valine are expressed in ω i and the values are expressed in percentage content (%), and are calculated according to the following formula:

[0086]

[0087] In the formula:

[0088] Ai——the peak area of L-leucine, L-isoleucine, and L-valine in the sample;

[0089] cs——the concentration of L-leucine, L-isoleucine, and L-valine control solutions, in nanomoles per milliliter (nmol / mL);

[0090] V——the constant volume of the sample solution, in milliliters (mL);

[0091] M i ——The molar mass of L-leucine, L-isoleucine, and L-valine in grams per mole (g / mol) (M(L-leucine,

[0092] L-isoleucine) = 131.17, M (L-valine) = 117.15);

[0093] f——dilution multiple;

[0094] As——peak area of ​​L-leucine, L-isoleucine, and L-valine control solutions;

[0095] m——Numerical value of sample mass, in g;

[0096] 10 9 ——conversion factor.

[0097] The test results are expressed as the arithmetic mean of the parallel determination results, and the results are rounded to three significant figures.

[0098] Total amino acid content: The total amino acid content is the sum of the contents of each monomer amino acid.

[0099] Precision: The absolute difference between two independent measurement results obtained under repeatability conditions should not be greater than 3% of the arithmetic mean.

[0100] If the centrifuge tube 3 is not improved, in order to avoid the influence of the instability of OPA-amino acid as much as possible, in the process of "determination of sample solution", 30 μL of tri(2-carboxyl)phosphine (TCEP) solution can be further added to the "mixed liquid 5 to be determined" obtained by gradually and quantitatively adding the sample solution, borate buffer, OPA solution and water into the centrifuge tube 3; by combining with the free radicals or active oxygen generated in the OPA reaction, OPA is prevented from being oxidized, thereby stabilizing the OPA-amino acid derivative; increasing the concentration of β-mercaptoethanol can improve the stability of the derivatization reagent; although this method can alleviate the influence of the instability of OPA-amino acid, excessive tri(2-carboxyl)phosphine (TCEP) solution may also interfere with chromatographic separation and detection, so it is necessary to select a suitable addition concentration to control its dosage under the premise of ensuring stability.

[0101] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for detecting instant free branched-chain amino acid monomers, characterized in that: Prepare water, acetonitrile, methanol, hydrochloric acid solution, acetic acid solution, sodium hydroxide solution, OPA solution, borate buffer solution, acetate buffer solution I, acetate buffer solution II, leucine reference substance, isoleucine reference substance, valine reference substance; high performance liquid chromatograph, liquid chromatography processor, chromatographic column, analytical balance and centrifuge tubes (3); the detection steps are successively "preparing the reference solution", "preparing the sample solution", "determining the sample solution and the reference solution", and "result calculation".

2. The detection method of instant branched-chain amino acid free monomers according to claim 1, characterized in that: The method of preparing the OPA solution immediately before use: Weigh 0.08 g of phthalaldehyde, dissolve it in 7 mL of boric acid buffer solution and 1 mL of acetonitrile, and add 125 μL of 3-mercaptopropionic acid, and mix well.

3. The detection method of instant branched-chain amino acid free monomers according to claim 1, characterized in that: The preparation method of the borate buffer solution: Weigh 24.7 g of boric acid, dissolve it in 800 mL of water, adjust the pH to 10.2 with sodium hydroxide solution, and dilute it to 1000 mL with water; the preparation method of the acetate buffer solution I: Weigh 6.0 g of sodium acetate, dissolve it in 4000 mL of water, add 800 μL of triethylamine and 24 mL of tetrahydrofuran, mix well, and adjust the pH to 7.2 with acetic acid solution; the preparation method of the acetate buffer solution II: Weigh 10.9 g of sodium acetate, dissolve it in 800 mL of water, and adjust the pH to 7.2 with acetic acid solution.

4. The detection method of instant branched-chain amino acid free monomers according to claim 1, characterized in that: The method of "preparing the reference solution": Weigh 0.1 g of the reference substance, dissolve it in water and make the volume up to 100 mL respectively; the method of "preparing the sample solution": Weigh 0.5 g of the sample, dissolve it in water and make the volume up to 250 mL respectively.

5. The detection method of instant branched-chain amino acid free monomers according to claim 1, characterized in that: The method of "determining the sample solution and the reference solution": Determination of the sample solution Precisely pipette 10 μL of the sample solution into the centrifuge tube (3), then continue to precisely add 50 μL of the borate buffer solution, precisely add 10 μL of the OPA solution, and add 330 μL of water to obtain the "mixed liquid to be determined (5)", mix the "mixed liquid to be determined (5)", and immediately precisely measure 40 μL and inject it into the high performance liquid chromatograph, and record the chromatogram. Determination of the reference solution Precisely measure 10 μL of the reference substance solution into the centrifuge tube (3), then continue to precisely add 50 μL of the borate buffer solution, precisely add 10 μL of the OPA solution, and add 330 μL of water to also obtain the "mixed liquid to be determined (5)", mix well, immediately precisely measure 40 μL and inject it into the high performance liquid chromatograph, and record the chromatogram.

6. The detection method of instant branched-chain amino acid free monomers according to claim 5, characterized in that: "Result calculation": The contents of L-leucine, L-isoleucine, and L-valine are in ω i calculated, and the values are expressed in percentage content (%), and calculated according to the following formula: Where: Ai—the peak areas of L-leucine, L-isoleucine, and L-valine in the sample; cs—the concentrations of the L-leucine, L-isoleucine, and L-valine reference solutions, in nanomoles per milliliter (nmol / mL); V—the volume of the sample solution made up, in milliliters (mL); M i ——The molar masses of L-leucine, L-isoleucine, and L-valine, in grams per mole (g / mol) (M(L-leucine, L-isoleucine) = 131.17, M(L-valine) = 117.15); f—the dilution factor; As—the peak areas of the L-leucine, L-isoleucine, and L-valine reference solutions; m—the numerical value of the sample mass, in grams (g); 10 9 ——conversion coefficient; The test results are expressed as the arithmetic mean of the parallel determination results, and the results are retained to three significant figures. Total amino acid content: The total amino acid content is the sum of the contents of each monomer amino acid; Precision: The absolute difference between two independent determination results obtained under repeatability conditions shall not be greater than 3% of the arithmetic mean.

7. A centrifuge tube for detecting instant branched-chain amino acid free monomers, characterized in that: The wall of the conical shell head (6) at the lower end of the centrifuge tube (3) is evenly hollowed out with liquid seepage holes (7). The inner wall surface of the conical shell head (6) is attached with a conical elastic silica gel film (8). The upper film contour (4) of the conical elastic silica gel film (8) is integrally connected with the inner wall of the upper end of the conical shell head (6) along the contour; a number of paddle pieces (2) are arranged in a circular array on the inner wall of the lower end of the cylindrical part (3a) of the centrifuge tube (3); it also includes a rotating cup (10) that cooperates with the improved centrifuge tube (3). The lower end of the rotating cup (10) is coaxially fixedly connected with a vertical motor output shaft (11) with a forward and reverse rotation function; pre-installed water (9) at 32°C to 38°C is pre-injected into the cup cavity inside the rotating cup (10). The inner wall of the upper end of the rotating cup (10) is provided with a piston sealing ring (13) along the wheel. The inner diameter of the piston sealing ring (13) is adapted to the outer diameter of the cylindrical part (3a) of the centrifuge tube (3); when the conical shell head (6) at the lower end of the centrifuge tube (3) is just completely inserted coaxially downward into the rotating cup (10), the liquid level of the pre-installed water (9) rises to the upper end position of the rotating cup (10), and the piston sealing ring (13) is hermetically sleeved on the outer wall of the lower end of the cylindrical part (3a) of the centrifuge tube (3), and a static friction force is formed between the piston sealing ring (13) and the centrifuge tube (3).

8. The working method of the centrifuge tube for detecting instant branched-chain amino acid free monomers according to claim 7, characterized in that: During the "determination of the sample solution" process, after gradually and quantitatively adding the sample solution, borate buffer solution, OPA solution and water into the centrifuge tube (3), the "mixed liquid to be determined (5)" is gathered inside the conical elastic silica gel film (8) at the bottom end of the centrifuge tube (3). Subsequently, immediately push down the centrifuge tube (3) forcefully by hand or tool to make the centrifuge tube (3) displace downward until the lower end of the centrifuge tube (3) is in limit contact with the bottom of the cup inside the rotating cup (10); after covering the centrifuge tube cap (14), immediately control the motor output shaft (11) to perform periodic alternating forward and reverse rotations; after the reaction is completed, immediately pull up the centrifuge tube (3) forcefully after opening the centrifuge tube cap (14) to make the centrifuge tube (3) displace upward relative to the rotating cup (10) to the initial position, and immediately quantitatively inject the reacted "mixed liquid to be determined (5)" into the liquid chromatograph by a pipette.

9. The detection method of instant branched-chain amino acid free monomer according to claim 5, characterized in that: During the "determination of the sample solution" process, 30 μL of tris(2-carboxy)phosphine (TCEP) solution is further added to the "mixed liquid to be determined (5)" obtained by gradually and quantitatively adding the sample solution, borate buffer solution, OPA solution and water into the centrifuge tube (3).

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