Detection Method and Apparatus for Free Monomers of Fast-Soluble Branched-Chain Amino Acids

Abstract

This invention discloses a method for detecting rapidly soluble branched-chain amino acid free monomers. The method involves preparing water, acetonitrile, methanol, hydrochloric acid solution, acetic acid solution, sodium hydroxide solution, OPA solution, borate buffer, acetate buffer I, acetate buffer II, leucine reference standard, isoleucine reference standard, and valine reference standard; a high-performance liquid chromatograph, a liquid chromatography processor, a chromatographic column, an analytical balance, and centrifuge tubes; the detection steps are sequentially: “preparation of reference solution,” “preparation of sample solution,” “determination of sample solution and reference solution,” and “result calculation.” This provides a highly efficient quantitative detection scheme for rapidly soluble branched-chain amino acid free monomers.

Description

Technical Field

[0001] This invention belongs to the field of amino acid detection. Background Technology

[0002] The free monomer content in instant branched-chain amino acid products is one of the important indicators for measuring their quality. By detecting the free monomer content, the purity, stability and efficacy of the product can be assessed, ensuring that the product meets relevant standards and regulations.

[0003] Understanding the content of free monomers in instant branched-chain amino acid (BCAA) products helps in developing personalized nutritional supplementation plans. For individuals who need BCAA supplementation, intake can be adjusted based on test results to meet the body's nutritional needs. Certain chronic diseases or metabolic abnormalities may affect the metabolism of BCAAs. By regularly testing the content of free monomers in instant BCAAs, these potential health problems can be detected in a timely manner, allowing time for early intervention and treatment.

[0004] In fields such as biomedical research, nutrition, and ecology, the detection of free amino acids is of great significance for understanding the state of life activities, exploring the mechanisms of disease, and guiding nutritional intake. The detection of free monomers of readily soluble branched-chain amino acids also helps to advance scientific research and applications in these fields.

[0005] This solution provides an efficient quantitative detection method for rapidly soluble branched-chain amino acid free monomers. Summary of the Invention

[0006] Purpose of the invention: In order to overcome the shortcomings of the existing technology, the present invention provides a method and device for detecting rapidly soluble branched-chain amino acid free monomers, and provides an efficient quantitative detection scheme for rapidly soluble branched-chain amino acid free monomers.

[0007] Technical Solution: To achieve the above objectives, the present invention provides a method for detecting rapidly soluble branched-chain amino acid free monomers, comprising: preparing water, acetonitrile, methanol, hydrochloric acid solution, acetic acid solution, sodium hydroxide solution, OPA solution, borate buffer, acetate buffer I, acetate buffer II, leucine reference standard, isoleucine reference standard, and valine reference standard; a high-performance liquid chromatograph, a liquid chromatography processor, a chromatographic column, an analytical balance, and centrifuge tubes; the detection steps are as follows: “preparing the reference solution”, “preparing the sample solution”, “measuring the sample solution and the reference solution”, and “calculating the results”.

[0008] Furthermore, the OPA solution is prepared immediately before use as follows: Weigh 0.08 g of o-phthalaldehyde, accurate to 0.0001 g, add 7 mL of borate slow-release solution and 1 mL of acetonitrile to dissolve it, add 125 μL of 3-mercaptopropionic acid, and mix well.

[0009] Furthermore, the borate buffer solution is prepared as follows: 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 with water to 1000 mL; the acetate buffer solution I is prepared as follows: 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 acetate buffer solution II is prepared as follows: 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.

[0010] Furthermore, the method for "preparing the control solution" is as follows: weigh 0.1 g of the reference standard, accurate to 0.0001 g, dissolve it in water, and dilute to 100 mL; the method for "preparing the sample solution" is as follows: weigh 0.5 g of the sample, accurate to 0.0001 g, dissolve it in water, and dilute to 250 mL.

[0011] Further, the method for "determining the sample solution and control solution":

[0012] Determination of sample solution

[0013] Accurately transfer 10 μL of the sample solution into centrifuge tube 3, accurately add 50 μL of borate buffer, accurately add 10 μL of OPA solution, and add 330 μL of water to obtain "mixed liquid to be tested 5". The primary amino acid in "mixed liquid to be tested 5" can react with o-phenylenedialdehyde (OPA) to generate OPA-amino acid. After mixing the mixed liquid to be tested 5 well, immediately accurately measure 40 μL and inject it into the liquid chromatograph and record the chromatogram.

[0014] Determination of control solution

[0015] Accurately measure 10 μL of the reference solution and place it in a centrifuge tube. Accurately add 50 μL of borate buffer, 10 μL of OPA solution, and 330 μL of water to obtain the "mixed liquid to be tested". Mix well and immediately accurately measure 40 μL and inject it into the liquid chromatograph. Record the chromatogram.

[0016] Furthermore, “Result Calculation”:

[0017] The content of L-leucine, L-isoleucine, and L-valine is based on The values ​​are expressed as percentages (%) and are calculated using the following formula:

[0018]

[0019] In the formula:

[0020] Ai — Peak areas of L-leucine, L-isoleucine, and L-valine in the sample;

[0021] cs—Concentration of L-leucine, L-isoleucine, and L-valine control solutions, in nanomoles per milliliter (nmol / mL).

[0022] V—The final volume of the sample solution, in milliliters (mL);

[0023] M i —Molar mass of L-leucine, L-isoleucine, and L-valine, in grams per mole (g / mol) (M(L-leucine, L-isoleucine, L-valine)

[0024] L-Isoleucine = 131.17, M (L-Valine) = 117.15).

[0025] f—Dilution factor;

[0026] As—peak areas of L-leucine, L-isoleucine, and L-valine control solutions;

[0027] m — the numerical value of the sample mass, in g;

[0028] 10 9 —Conversion factor.

[0029] The test results are expressed as the arithmetic mean of parallel measurements, and the results are retained to three significant figures.

[0030] Total amino acid content: Total amino acid content is the sum of the amino acid content of each monomer;

[0031] Precision: The absolute difference between two independent measurements obtained under repeatability conditions should not exceed 3% of the arithmetic mean.

[0032] Furthermore, the conical head at the lower end of the centrifuge tube has uniformly perforated drainage holes on its wall. A conical elastic silicone film is attached to the inner wall of the conical head, with the upper contour of the film integrally connected to the inner wall of the upper end of the conical head along the contour. Several paddles are arranged in a circular pattern on the lower inner wall of the cylindrical portion of the centrifuge tube. A rotating cup that cooperates with the improved centrifuge tube is also included. A vertical motor output shaft with forward and reverse rotation functions is coaxially fixed to the lower end of the rotating cup. Pre-filled water at 32°C to 38°C is injected into the inner cavity of the rotating cup. A piston sealing ring is installed along the upper inner wall of the rotating cup, with the inner diameter of the piston sealing ring matching the outer diameter of the cylindrical portion of the centrifuge tube. When the conical head at the lower end of the centrifuge tube is fully coaxially inserted into the rotating cup, the pre-filled water level rises to the upper position of the rotating cup, and the piston sealing ring seals against the lower outer wall of the cylindrical portion of the centrifuge tube, creating static friction between the piston sealing ring and the centrifuge tube.

[0033] Furthermore, in the "determination of sample solution" process, the sample solution, borate buffer, OPA solution and water are gradually and quantitatively added to the centrifuge tube. The resulting "mixed liquid to be measured" accumulates inside the conical elastic silicone membrane at the bottom of the centrifuge tube. Then, the centrifuge tube is immediately and forcibly pushed downward by hand or tool, causing it to move downward until the lower end of the centrifuge tube contacts the bottom of the rotating cup. After the centrifuge tube is capped, the motor output shaft is immediately controlled to periodically alternate forward and reverse rotation, with the periodic alternation time controlled within 5 seconds. After the reaction is completed, the centrifuge tube cap is opened and the centrifuge tube is immediately and forcibly pulled upward, causing it to move upward relative to the rotating cup to the initial position. The reacted "mixed liquid to be measured" is then quantitatively injected into the liquid chromatograph using a pipette.

[0034] Furthermore, during the "determination of the sample solution", 30 μL of tris(2-carboxy)phosphine (TCEP) solution was added to the "mixed liquid to be tested" obtained by gradually and quantitatively adding the sample solution, borate buffer, OPA solution and water to the centrifuge tube.

[0035] Beneficial effects: This invention provides a highly efficient quantitative detection scheme for rapidly soluble branched-chain amino acid free monomers. At the same time, in the process of "determination of sample solution", the mixing and reaction of the liquid to be tested can be completed rapidly in a short time, avoiding the problems of inaccurate detection caused by insufficient reaction (insufficient mixing) and excessive reaction time. Attached Figure Description

[0036] Figure 1 The main reaction formulas in the detection process of this scheme;

[0037] Figure 2 This is a flowchart of the detection method;

[0038] Figure 3 A schematic diagram of the newly designed centrifuge tubes and spinneret;

[0039] Figure 4 Schematic diagrams of the two configurations of the newly designed centrifuge tubes and rotating cups. Detailed Implementation

[0040] The invention will now be further described with reference to the accompanying drawings.

[0041] In the presence of a thiol reagent, primary amino acids can react with o-phthalaldehyde (OPA) to form OPA-amino acids. After separation by reversed-phase high-performance liquid chromatography (RP-HPLC), the OPA-amino acid derivative is detected at 338 nm using a UV detector. Within a certain concentration range (25 pmol–2500 pmol), the absorbance is directly proportional to the amino acid concentration. Quantification is performed using the external standard method. In the presence of 3-mercaptopropionic acid, a simplified reaction formula for the reaction of amino acids (represented by the general formula RCH(NH2)COOH) with OPA to form OPA-amino acid derivatives can be expressed as follows: Figure 1 As shown, in the above reaction formula, R represents the side chain group of an amino acid; the actual product is an isoindole derivative, which is simplified as "OPA - RCH (NH - CO - CH2 - S -CH2CH2COOH)".

[0042] Based on the above principles, the following detection scheme is designed in this case, and the flowchart is as follows: Figure 2 As shown:

[0043] Reagents and materials:

[0044] Water: GB / T 6682, Grade I water.

[0045] Acetonitrile: chromatographic grade.

[0046] Methanol: chromatographic grade.

[0047] Hydrochloric acid solution: 0.1 mol / L.

[0048] Acetic acid solution: Weigh 2.0 g of acetic acid and dissolve it in 98 mL of water.

[0049] Sodium hydroxide solution: Weigh 40.0 g of sodium hydroxide and dissolve it in 60 mL of water.

[0050] OPA solution: Weigh 0.08 g of o-phthalaldehyde, accurate to 0.0001 g, add 7 mL of borate slow salt solution and 1 mL of acetonitrile to dissolve, add 125 μL of 3-mercaptopropionic acid, mix well, and prepare fresh before use.

[0051] Borate buffer: 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 with water to 1000 mL.

[0052] Acetate buffer 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.

[0053] Acetate buffer 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.

[0054] Leucine reference standard (C6H) 13 NO2 (CAS No.: 61-90-5): Purity ≥ 99.0%, or a standard substance that has been certified by the state and granted a standard substance certificate.

[0055] Isoleucine reference standard (C6H) 13 NO2 (CAS No.: 73-32-5): Purity ≥ 99.0%, or a standard substance that has been certified by the state and granted a standard substance certificate.

[0056] Valine reference standard (C5H) 11 NO2 (CAS No.: 72-18-4): Purity ≥ 99.0%, or a standard substance that has been certified by the state and granted a standard substance certificate.

[0057] Instruments and equipment:

[0058] High performance liquid chromatograph: equipped with a UV detector and an autosampler.

[0059] Liquid chromatography processor: chemical workstation.

[0060] Chromatographic column: C18 (octadecylsilane-bonded silica gel as packing material), 250 mm × 4.6 mm, 5 μm, or equivalent.

[0061] Analytical balance: sensitivity 0.0001 g.

[0062] Centrifuge tube 3: 1.5 mL.

[0063] step:

[0064] Preparation of control solutions: Weigh 0.1 g of the reference standard, accurate to 0.0001 g, dissolve in water and dilute to 100 mL.

[0065] Preparation of sample solution: Weigh 0.5 g of sample, accurate to 0.0001 g, dissolve in water and dilute to 250 mL.

[0066] Instrument reference conditions:

[0067] Detection wavelength: 338 nm.

[0068] Temperature: 35 ℃.

[0069] Mobile phase A: Acetate buffer I.

[0070] Mobile phase B: Acetate buffer II + acetonitrile + methanol = 800 + 1400 + 1800.

[0071] The gradient elution procedure for the mobile phase is shown in Table A.1.

[0072] Table A.1 Gradient elution procedure

[0073]

[0074] Determination of sample solution

[0075] Accurately transfer 10 μL of the sample solution into centrifuge tube 3, accurately add 50 μL of borate buffer, accurately add 10 μL of OPA solution, and add 330 μL of water to obtain "mixed liquid to be tested 5". The primary amino acid in "mixed liquid to be tested 5" can react with o-phenylenedialdehyde (OPA) to generate OPA-amino acid. After mixing the mixed liquid to be tested 5 well, immediately accurately measure 40 μL and inject it into the liquid chromatograph and record the chromatogram.

[0076] Determination of control solution

[0077] Accurately measure 10 μL of the reference solution and place it in centrifuge tube 3. Accurately add 50 μL of borate buffer, 10 μL of OPA solution, and 330 μL of water to obtain "mixed liquid to be tested 5". Mix well and immediately accurately measure 40 μL and inject it into the liquid chromatograph. Record the chromatogram.

[0078] Note: OPA-amino acids are unstable and should be analyzed immediately after the derivatization reaction is complete.

[0079] During the "determination of sample solution", the OPA-amino acid obtained by the reaction of the mixed liquid 5 to be tested in centrifuge tube 3 is unstable. The mixing and reaction of the mixed liquid 5 to be tested need to be completed quickly in a short time. Whether the reaction is insufficient (insufficient mixing) or the reaction time is too long, it will cause inaccurate detection. Therefore, it is necessary to implement a more efficient and rapid mixing reaction for the mixed liquid 5 to be tested in centrifuge tube 3.

[0080] like Figure 3As shown, the lower end of a common centrifuge tube 3 has a conical head 6 structure. In the above-mentioned "determination of sample solution", the total amount of the sample solution added to the centrifuge tube 3 is only 400 μL of the test liquid 5 to be mixed. The test liquid 5 added to the centrifuge tube 3 will eventually accumulate in the conical head 6. Due to the small liquid volume, it is not conducive to manual or machine mixing. If the centrifuge tube 3 is rotated alternately in both forward and reverse directions, the rotational linear velocity of the test liquid 5 is low because it accumulates near the axis of the centrifuge tube 3. It is difficult to fully mix the test liquid 5 in a very short time by alternating forward and reverse rotation of the centrifuge tube 3 using the liquid's own inertia and the friction of the inner wall. In order to enable the test liquid 5 in the centrifuge tube 3 to be efficiently mixed and reacted in a shorter time, the centrifuge tube 3 is improved as follows:

[0081] The newly designed conical head 6 has uniformly perforated drainage holes 7 on its wall. A conical elastic silicone film 8, 0.5 mm thick, is attached to the inner wall of the conical head 6. The upper contour 4 of the conical elastic silicone film 8 is integrally heat-sealed with the upper inner wall of the conical head 6 along the contour. Several paddles 2 are arranged in a circular pattern on the lower inner wall of the cylindrical portion 3a of the centrifuge tube 3. A rotating cup that cooperates with the improved centrifuge tube 3 is also included. 10. A vertical motor output shaft 11 with forward and reverse rotation function is coaxially fixedly connected to the lower end of the rotating cup 10; the inner cavity of the rotating cup 10 is pre-filled with water 9 at a temperature of 32°C to 38°C; a silicone piston seal ring 13 is provided along the upper inner wall of the rotating cup 10, and 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; the initial state is when the conical head 6 at the lower end of the centrifuge tube 3 is just fully coaxially inserted downwards into the rotating cup 10. Figure 4 As shown on the left, the pre-filled water 9 rises to the upper position of the rotating cup 10, and the piston sealing ring 13 is sealed to the lower outer wall of the column part 3a of the centrifuge tube 3. Static friction is formed between the piston sealing ring 13 and the centrifuge tube 3. The maximum static friction can be adjusted in the design stage by the material properties and the tightness of the fit.

[0082] The highly efficient and rapid mixing reaction principle and working process of the newly designed centrifuge tube 3:

[0083] In the initial state, the conical head 6 at the lower end of the centrifuge tube 3 is inserted into the rotating cup 10 with its axis pointing downwards. Figure 4As shown in the left figure, the pre-filled water 9 is at the upper end of the rotating cup 10, and the piston sealing ring 13 is sealed to the lower outer wall of the column part 3a of the centrifuge tube 3. During the "determination of the sample solution", the "mixed liquid to be measured 5" obtained by gradually and quantitatively adding the sample solution, borate buffer, OPA solution and water into the centrifuge tube 3 accumulates inside the conical elastic silicone film 8 at the bottom of the centrifuge tube 3. Then, the centrifuge tube 3 is immediately and forcibly pushed downward by hand or tool to move the centrifuge tube 3 downward until the centrifuge tube is fully displaced. 3. The lower end of the centrifuge tube 3 is in contact with the bottom of the rotating cup 10. During the downward displacement of the centrifuge tube 3, the pre-filled water 9 in the rotating cup 10 is squeezed, causing the pre-filled water 9 that was originally in the rotating cup 10 to be squeezed through the evenly perforated seepage holes 7 on the wall of the conical head 6 and squeezed into the space between the inner wall of the conical head 6 and the conical elastic silicone film 8. This causes the conical elastic silicone film 8 to deform upward under the pressure of the pre-filled water 9, until the original conical elastic silicone film 8 with its tip pointing downward is deformed upward to its tip pointing upward. Figure 4 As shown in the right figure, at this time, the "mixed liquid 5 to be measured" is gathered in the annular water groove 5a between the conical elastic silicone film 8 and the cylindrical part 3a of the centrifuge tube 3. The "mixed liquid 5 to be measured" in the annular water groove 5a is further away 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 between the piston seal ring 13 and the centrifuge tube 3 keeps the centrifuge tube 3 and the rotating cup 10 in a synchronized state. Figure 4 The image on the right;

[0084] After the above process is completed, the centrifuge tube cap 14 is closed, and the motor output shaft 11 is immediately controlled to periodically alternate forward and reverse rotation, so that the centrifuge tube 3 and the rotating cup 10 periodically alternate forward and reverse rotation. The "mixed liquid 5 to be tested" in the annular water ditch 5a, under its own inertia and the friction, shearing and stirring of the inner wall of the annular water ditch 5a and several paddles 2, rolls, surges and tears back and forth in the circumferential direction, so that the primary amino acids and o-phthalaldehyde OPA in the "mixed liquid 5 to be tested" react rapidly and fully in a short time under the environment of rolling, surging and tearing, to generate OPA-amino acids; the periodic alternating forward and reverse rotation time of the motor output shaft 11 is controlled within 5 seconds;

[0085] After the reaction is complete, the reacted "mixture 5" needs to be injected quantitatively into the liquid chromatograph immediately using a pipette. However, the "mixture 5" accumulated in the annular groove 5a is not easily accessible by pipette. Therefore, after opening the centrifuge tube cap 14, the centrifuge tube 3 needs to be forcibly pulled upwards to move it relative to the rotating cup 10 to its initial position. During the upward movement of the centrifuge tube 3 relative to the rotating cup 10, a negative pressure is generated on the lower side of the conical elastic silicone film 8. Under the action of the negative pressure, the conical elastic silicone film 8 reattaches to the inner wall of the conical head 6. Figure 4 The left image shows the completely reacted "mixture 5" re-aggregating inside the conical elastic silicone film 8 at the bottom of centrifuge tube 3, as shown in the left image. Figure 4 It is positioned on the left side, making it easier to access the pipette.

[0086] Result Calculation

[0087] The content of L-leucine, L-isoleucine, and L-valine is based on The values ​​are expressed as percentages (%) and are calculated using the following formula:

[0088]

[0089] In the formula:

[0090] Ai — Peak areas of L-leucine, L-isoleucine, and L-valine in the sample;

[0091] cs—Concentration of L-leucine, L-isoleucine, and L-valine control solutions, in nanomoles per milliliter (nmol / mL).

[0092] V—The final volume of the sample solution, in milliliters (mL);

[0093] M i —Molar mass of L-leucine, L-isoleucine, and L-valine, in grams per mole (g / mol) (M(L-leucine, L-isoleucine, L-valine)

[0094] L-Isoleucine = 131.17, M (L-Valine) = 117.15).

[0095] f—Dilution factor;

[0096] As—peak areas of L-leucine, L-isoleucine, and L-valine control solutions;

[0097] m — the numerical value of the sample mass, in g;

[0098] 10 9 —Conversion factor.

[0099] The test results are expressed as the arithmetic mean of parallel measurements, and the results are retained to three significant figures.

[0100] Total amino acid content: Total amino acid content is the sum of the amino acid content of each monomer.

[0101] Precision: The absolute difference between two independent measurements obtained under repeatability conditions should not exceed 3% of the arithmetic mean.

[0102] If centrifuge tube 3 is not improved, to minimize the impact of OPA-amino acid instability, during the "sample solution determination" process, 30 μL of tris(2-carboxy)phosphine (TCEP) solution can be added to the "mixed liquid to be measured 5" obtained by gradually and quantitatively adding the sample solution, borate buffer, OPA solution, and water to centrifuge tube 3. This will prevent OPA from being oxidized by binding with free radicals or reactive oxygen species generated during the OPA reaction, thereby stabilizing the OPA-amino acid derivative. Increasing the concentration of β-mercaptoethanol can improve the stability of the derivatizing reagent. Although this method can alleviate the impact of OPA-amino acid instability, excessive tris(2-carboxy)phosphine (TCEP) solution may also interfere with chromatographic separation and detection. Therefore, it is necessary to select an appropriate addition concentration to control its amount while ensuring stability.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A centrifuge tube for detecting rapidly soluble branched-chain amino acid free monomers, characterized in that: The centrifuge tube (3) has uniformly perforated drainage holes (7) on the wall of the conical head (6) at the lower end. A conical elastic silicone film (8) is attached to the inner wall of the conical head (6). The upper contour (4) of the conical elastic silicone film (8) is integrally connected to the upper inner wall of the conical head (6) along the contour. Several paddles (2) are arranged in a circular pattern on the lower inner wall 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 to a vertical motor output shaft (11) with forward and reverse rotation functions. The rotating cup (10) contains... The side cup cavity is pre-filled with pre-filled water (9) at 32°C to 38°C. The upper inner wall of the rotating cup (10) is provided with a piston sealing ring (13) along the contour. 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 head (6) at the lower end of the centrifuge tube (3) is just completely coaxially inserted into the rotating cup (10), the liquid level of the pre-filled water (9) rises to the upper position of the rotating cup (10), and the piston sealing ring (13) is sealed and sleeved with the lower outer wall of the cylindrical part (3a) of the centrifuge tube (3). Static friction is formed between the piston sealing ring (13) and the centrifuge tube (3). During the "determination of the sample solution", the "mixed liquid to be tested (5)" obtained by gradually and quantitatively adding the sample solution, borate buffer, OPA solution and water into the centrifuge tube (3) is collected inside the conical elastic silicone film (8) at the bottom of the centrifuge tube (3). Then, the centrifuge tube (3) is immediately forced downward by hand or tool to move downward until the lower end of the centrifuge tube (3) contacts the bottom of the rotating cup (10). During the downward movement of the centrifuge tube (3), the pre-filled water (9) in the rotating cup (10) is squeezed, and the pre-filled water (9) that was originally in the rotating cup (10) is squeezed through the uniformly hollowed permeation holes (7) on the wall of the conical head (6) and squeezed into the space between the inner wall of the conical head (6) and the conical elastic silicone film (8) under the pressure of the pre-filled water (9). This causes the conical elastic silicone film (8) to deform upward under the pressure of the pre-filled water (9) until the conical elastic silicone film (8) that was originally downward is deformed upward and the tip is upward.

2. The method for operating a centrifuge tube for detecting rapidly soluble branched-chain amino acid free monomers according to claim 1, characterized in that: During the "determination of sample solution", the sample solution, borate buffer, OPA solution and water are gradually added quantitatively to the centrifuge tube (3) to obtain the "mixed liquid to be measured (5)" which gathers inside the conical elastic silicone film (8) at the bottom of the centrifuge tube (3). Then, the centrifuge tube (3) is immediately forced downward by hand or tool to move downward until the lower end of the centrifuge tube (3) contacts the bottom of the rotating cup (10). After the centrifuge tube cap (14) is closed, the motor output shaft (11) is immediately controlled to alternately rotate in a periodic forward and reverse direction. After the reaction is completed, the centrifuge tube cap (14) is opened and the centrifuge tube (3) is immediately forced upward to move upward relative to the rotating cup (10) to the initial position. The reacted "mixed liquid to be measured (5)" is then quantitatively injected into the liquid chromatograph using a pipette.

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