A capillary electrophoresis method for serum amino acid analysis

Abstract

This invention provides a capillary electrophoresis analysis method for serum amino acids, comprising the following steps: S1 preparation of running buffer; S2 selection and pretreatment of a quartz capillary column; S3 electrophoresis analysis conditions; S4 plotting a quasi-curve; S5 sample derivatization; and S6 quantitative analysis. This method is the first to employ... α‑ Cyclodextrin, β‑ Cyclodextrin and c‑ The cyclodextrin mixture synergistically regulates amino acid migration behavior, expands the migration time window, effectively improves the separation resolution of structurally similar amino acids, and significantly enhances separation efficiency and chromatographic clarity. The addition of sodium taurocholate, along with pH adjustment, forms a weakly alkaline buffer system with moderate conductivity and stable migration behavior, which is beneficial for the separation and detection of FITC-derived amino acids and optimizes the uniformity of migration time distribution. This invention is applicable to clinical sample analysis, disease biomarker screening, and other scenarios, possessing good versatility, stability, and promotional value.

Description

Technical Field

[0001] This invention relates to the field of biochemical analysis technology, specifically to a capillary electrophoresis analysis method for serum amino acids. Background Technology

[0002] Amino acids are the basic building blocks of proteins, and proteins are the material basis of life; therefore, amino acids play a vital role in life processes. Clinically, amino acid determination is not only of great value in the diagnosis and treatment of metabolic diseases, kidney diseases, and liver diseases, but has also received widespread attention in recent years for evaluating patients' nutritional status. Capillary electrophoresis is a high-performance liquid chromatography (HPLC) separation technique. Due to its advantages of high efficiency, speed, sensitivity, and low consumption, it has been rapidly adopted for amino acid analysis. Most of the 20 amino acids that make up human proteins do not have ultraviolet absorption peaks or produce fluorescence, and derivatization is generally required. Current methods for detecting amino acids using capillary electrophoresis are relatively mature, but in practice, problems such as low separation efficiency, severe peak overlap, and poor adaptability still exist.

[0003] Therefore, there is an urgent need in this field for a capillary electrophoresis detection method that can improve separation efficiency and enhance the versatility and stability of the method. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a serum amino acid capillary electrophoresis analysis method with high separation efficiency, strong method versatility and stability. To solve the above technical problem, this invention provides the following technical solution:

[0005] This invention provides a capillary electrophoresis analysis method for serum amino acids, comprising the following steps:

[0006] S1 running buffer preparation: The running buffer aqueous solution contains 80-100 mmol / L sodium tetraborate decahydrate and 1-3 mmol / L sodium taurocholate. α- Cyclodextrin 70~90 mmol / L β- Cyclodextrin 10~30 mmol / L γ- Add 5-20 mmol / L cyclodextrin, adjust the pH to 9.0, filter and set aside;

[0007] S2 Quartz Capillary Column Selection and Pretreatment: Uncoated quartz capillary columns were selected. Before use, the columns were rinsed sequentially with NaOH solution, deionized water, and running buffer. Before each injection, the columns were rinsed with deionized water and running buffer, respectively.

[0008] S3 electrophoresis analysis conditions: (1) Injection method: pressure injection method; (2) Operating conditions: separate voltage, separation current, capillary temperature and electrophoresis time are set respectively; (3) Detector: fluorescence detector;

[0009] S4. Plotting the quasi-curve: Five groups of standard amino acid solutions with different concentrations (20, 1 ... μ M, 50 μ M, 100 μ M, 250 μ M and 500 μ M, perform the electrophoretic analysis conditions described in S3 sequentially, record the electrophoretic peak area corresponding to each standard sample, plot the standard curve with amino acid concentration as the abscissa and peak area as the ordinate, and establish a linear or nonlinear functional relationship between amino acid concentration and peak area.

[0010] S5 Sample derivatization: The test sample was derivatized using an acetonitrile solution of fluorescein isothiocyanate with a concentration of 1~3 mmol / L, and the solvent of the solution was the same as that of the test sample;

[0011] S6 Quantitative Analysis:

[0012] The derivatized test sample is separated and detected according to the electrophoretic analysis conditions in S3 to obtain the electrophoretic peak diagram of the test sample. The peak area value of each peak is extracted and substituted into the function relationship constructed in step S4 to calculate the amino acid concentration value in the test sample.

[0013] Furthermore, in S1, the filtration process uses a strength of 0.2~0.6. μ microporous needle filter;

[0014] Furthermore, in S2, the concentration of the NaOH solution is 0.5~2M, and the rinsing time is 20~60min;

[0015] Furthermore, in S2, the deionized water rinsing time is 1~10 min;

[0016] Furthermore, in S2, the rinsing time of the running buffer is 10-15 minutes;

[0017] Furthermore, in S2, before each injection, the sample is rinsed with deionized water and running buffer for 1-3 minutes.

[0018] Furthermore, in S3, a pressure injection method is used, with an injection pressure of 0.1~0.5 psi and an injection time of 1~5s;

[0019] Furthermore, in S3, the operating conditions are set as follows: separation voltage 15~20 kV, separation current 0.1~0.3 kV. μ A. Capillary temperature 20~30℃, electrophoresis time 20~60min

[0020] Furthermore, in S3, the excitation wavelength of the fluorescence detector is 485 nm and the emission wavelength is 520 nm;

[0021] Furthermore, in S4, the standard amino acid solution is prepared as a mixed standard solution using 20 amino acid standard samples;

[0022] Furthermore, in S4, the functional relationships between the amino acid concentration and the peak area are as follows:

[0023] Leucine y=1690x, R 2 =0.999;

[0024] Tryptophan y = 519.2x - 4196, R 2 =0.997;

[0025] Isoleucine y = 1828x - 1906, R 2 =0.998;

[0026] Phenylalanine y = 2513.3x - 23845, R 2 =0.9983;

[0027] Methionine y = 3144x - 20346, R 2 =0.9937;

[0028] Valine y = 1026.1x + 5227.4, R 2 =0.9969;

[0029] Aspartic acid y = 2214.1x - 5664.8, R 2 =0.9964;

[0030] Proline y = 2077.9x + 30996, R 2 =0.9953;

[0031] Lysine y = 126.62x - 729.31, R 2 =0.9952;

[0032] Serine y = 261.76x - 2742.2, R 2 =0.999;

[0033] Threonine y = 81.319x + 886.3, R 2 =0.9978;

[0034] Histidine y = 310.15x - 5629.9, R 2 =0.9988;

[0035] Tyrosine y = 500.42x - 136.57, R 2 =0.9976;

[0036] Alanine y = 172.08x - 3774.2, R 2 =0.9972;

[0037] Glutamine y = 103.91x + 5815.3, R 2 =0.9981;

[0038] Glycine y = 322.73x + 3896.9, R 2 =0.9975;

[0039] Glutamic acid y = 378.93x - 4742.8, R 2 =0.9978;

[0040] Asparagine y = 51.599x - 892.7, R 2 =0.9991;

[0041] Cysteine ​​y = 318.38x - 77.129, R 2 =0.998;

[0042] Arginine y = 384.47x + 7.6703, R 2 =0.9984;

[0043] Furthermore, in S5, the sample derivatization step includes: taking the sample to be tested and adding 1.2 times its volume of 1~3 mmol / L fluorescein isothiocyanate acetonitrile solution, reacting in a water bath at 20~40℃ for 40 min~2 h to complete the fluorescent labeling of amino acids, then filtering with a 0.2~0.6 μm microporous needle filter, directly injecting the sample or storing it at -30~-10℃ for later use;

[0044] Compared with the prior art, the present invention has the following advantages:

[0045] 1. This invention provides a capillary electrophoresis analysis method for separating amino acids in serum and its running buffer, solving the problems of low separation efficiency, severe peak overlap, and poor adaptability of existing methods. This method is the first to utilize... α- Cyclodextrin, β- Cyclodextrin and γ- Cyclodextrin mixtures synergistically regulate amino acid migration behavior, expand the migration time window, effectively improve the separation resolution of structurally similar amino acids, and significantly enhance the separation degree and chromatographic clarity.

[0046] 2. The buffer solution of this invention is based on sodium borate, with the addition of a certain amount of sodium taurocholate, and the pH is adjusted to form a weakly alkaline buffer system with moderate conductivity and stable migration behavior, which is beneficial for the separation and detection of FITC-derived amino acids and optimizes the uniformity of migration time distribution.

[0047] 3. Electrophoresis parameters are set to constant temperature and pressure. Combined with fluorescence detection and sample derivatization, more than 20 amino acids in serum can be efficiently separated within 30 minutes. Compared with traditional methods using only a single cyclodextrin, this invention shows significant advantages in separating amino acids with similar structures (such as leucine / isoleucine), and is suitable for clinical sample analysis, disease biomarker screening, and other scenarios, possessing good versatility, stability, and promotional value.

[0048] 4. Introduction of sodium taurocholate as a separation aid: Adding specific sodium taurocholate to the buffer solution allows it to exert its interface regulation and decontamination effects, effectively reducing the adsorption of proteins or impurities in the serum matrix onto the capillary inner wall, further improving peak shape and enhancing the reproducibility and anti-interference ability of the method. Attached Figure Description

[0049] Figure 1 This is a standard curve of leucine in a mixed standard solution;

[0050] Figure 2 This is a standard curve of tryptophan in a mixed standard solution;

[0051] Figure 3 This is a standard curve of isoleucine in a mixed standard solution;

[0052] Figure 4 This is a standard curve of phenylalanine in a mixed standard solution;

[0053] Figure 5 This is a standard curve of methionine in a mixed standard solution;

[0054] Figure 6 This is a standard curve of valine in a mixed standard solution;

[0055] Figure 7 This is a standard curve of aspartic acid in a mixed standard solution.

[0056] Figure 8 This is a standard curve of proline in a mixed standard solution;

[0057] Figure 9 This is a standard curve of lysine in a mixed standard solution;

[0058] Figure 10 This is a standard curve of serine in a mixed standard solution;

[0059] Figure 11 This is a standard curve of threonine in a mixed standard solution;

[0060] Figure 12 This is a standard curve of histidine in a mixed standard solution.

[0061] Figure 13 This is a standard curve of tyrosine in a mixed standard solution;

[0062] Figure 14 This is a standard curve of alanine in a mixed standard solution;

[0063] Figure 15 This is a standard curve of glutamine in a mixed standard solution;

[0064] Figure 16 This is a standard curve of glycine in a mixed standard solution;

[0065] Figure 17 This is a standard curve of glutamic acid in a mixed standard solution;

[0066] Figure 18 The standard curve of asparagine in the mixed standard solution;

[0067] Figure 19 This is a standard curve of cysteine ​​in a mixed standard solution;

[0068] Figure 20 A standard curve of arginine in a mixed standard solution;

[0069] Figure 21 The detection graph shows the use of the buffer solution of this invention as a separation reagent;

[0070] Figure 22 The detection graph is for the separation reagent without added cyclodextrin;

[0071] Figure 23 Add only α - 、β - Detection diagram of the separation reagent for cyclodextrin;

[0072] Figure 24 The detection graph shows the use of cyclodextrin in a different proportion than the reagent used in the invention as the separating reagent. Detailed Implementation

[0073] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0074] (I) Implementation Examples

[0075] Preparation of S1 basal buffer (running buffer): Sodium tetraborate decahydrate (Borax) 90 mmol / L, sodium taurocholate 2 mmol / L. α -Cyclodextrin 80 mmol / L, β -Cyclodextrin 20 mmol / L, γ - Cyclodextrin 10 mmol / L; adjust pH to 9.0 (using 1M NaOH or HCl). After the solution is prepared, filter it through a 0.22 μm microporous membrane to avoid particles affecting the stability of the electrophoresis system.

[0076] S2 capillary column pretreatment: Use uncoated quartz capillaries with an inner diameter of 25 μm and a total length of 87 cm; before first use, rinse sequentially: 1 M NaOH solution for 30 minutes, deionized water for 5 minutes, and running buffer for 10-15 minutes; repeat the following steps before each injection: rinse with deionized water for 2 minutes and rinse with running buffer for 2 minutes.

[0077] S3 electrophoresis analysis conditions: (1) Injection method: pressure injection, injection pressure: 0.3 psi; injection time: 3 seconds; (2) Operating conditions: separation voltage: 15–20 kV, separation current: 0.2 μ A. Capillary temperature: constant at 25°C; total electrophoresis time: approximately 30 minutes; detection system: used to detect FITC-derived amino acids, fluorescence detector, excitation wavelength 485 nm, emission wavelength 520 nm;

[0078] S4. Plotting the quasi-curve: Five groups of standard amino acid solutions with different concentrations (20, 1 ... μ M, 50 μ M, 100 μ M, 250 μ M and 500 μ M, perform the electrophoretic analysis according to the conditions described in S3, and record the peak area of ​​each standard sample. Plot a standard curve with amino acid concentration on the x-axis and peak area on the y-axis to establish a linear or nonlinear functional relationship between amino acid concentration and peak area. The standard amino acid solution is a mixed standard solution prepared from 20 amino acid standard samples. The standard curve of the mixed standard solution is shown below. Figures 1-20 ;

[0079] The functional relationships between the amino acid concentration and the peak area are as follows:

[0080] Leucine y=1690x, R 2 =0.999;

[0081] Tryptophan y = 519.2x - 4196, R 2 =0.997;

[0082] Isoleucine y = 1828x - 1906, R 2 =0.998;

[0083] Phenylalanine y = 2513.3x - 23845, R 2 =0.9983;

[0084] Methionine y = 3144x - 20346, R 2 =0.9937;

[0085] Valine y = 1026.1x + 5227.4, R 2 =0.9969;

[0086] Aspartic acid y = 2214.1x - 5664.8, R 2 =0.9964;

[0087] Proline y = 2077.9x + 30996, R 2 =0.9953;

[0088] Lysine y = 126.62x - 729.31, R 2 =0.9952;

[0089] Serine y = 261.76x - 2742.2, R 2 =0.999;

[0090] Threonine y = 81.319x + 886.3, R 2 =0.9978;

[0091] Histidine y = 310.15x - 5629.9, R 2 =0.9988;

[0092] Tyrosine y = 500.42x - 136.57, R 2 =0.9976;

[0093] Alanine y = 172.08x - 3774.2, R 2 =0.9972;

[0094] Glutamine y = 103.91x + 5815.3, R 2 =0.9981;

[0095] Glycine y = 322.73x + 3896.9, R 2 =0.9975;

[0096] Glutamic acid y = 378.93x - 4742.8, R 2 =0.9978;

[0097] Asparagine y = 51.599x - 892.7, R 2 =0.9991;

[0098] Cysteine ​​y = 318.38x - 77.129, R 2 =0.998;

[0099] Arginine y = 384.47x + 7.6703, R 2 =0.9984;

[0100] S5 sample derivatization: Take 1 mL of serum acetonitrile treatment solution, add 1.2 mL of fluorescein isothiocyanate (FITC) acetonitrile solution (2 mmol / L), react in a 30℃ water bath for 1 hour to complete the fluorescent labeling of amino acids; the derivatized sample can be directly used for capillary electrophoresis or stored at -20℃ for later use.

[0101] S6 Quantitative Analysis: The derivatized sample to be tested is separated and detected according to the electrophoretic analysis conditions in S3 to obtain the electrophoretic peak diagram of the sample to be tested. The peak area value of each peak is extracted and substituted into the function relationship constructed in step S4 to calculate the amino acid concentration value in the sample to be tested.

[0102] Experimental Results: Under the above conditions, 20 common free amino acids (such as leucine, isoleucine, glutamic acid, lysine, etc.) can be completely separated within 30 minutes; the peak shape is symmetrical and the migration time is stable; the separation degree is better than that of the traditional monocyclic dextrin system, especially with structurally similar amino acids (such as Leu / Ile, Val / Thr). See also Figure 21 Using the reagents from the examples ( α -、 β -、 γ - Cyclodextrins: Three cyclodextrins in a fixed ratio (80:20:10 mmol / L) can successfully isolate 20 amino acids, including: 1. Arginine; 2. Lysine; 3. Leucine; 4. Isoleucine; 5. Methionine; 6. Phenylalanine; 7. Tryptophan; 8. Tyrosine; 9. Histidine; 10. Cysteine; 11. Valine; 12. Glutamine; 13. Proline; 14. Asparagine; 15. Threonine; 16. Glycine; 17. Serine; 18. Alanine; 19. Glutamic acid; 20. Aspartic acid.

[0103] (ii) Comparative Example

[0104] Comparative Example 1

[0105] Experimental methods:

[0106] Preparation of S1 basal buffer (running buffer): Sodium tetraborate decahydrate (Borax) 90 mmol / L, sodium taurocholate 2 mmol / L, adjust pH to 9.0 (using 1M NaOH or HCl). After the solution is prepared, use 0.22... μ Microporous membrane filtration is used to prevent particles from affecting the stability of the electrophoresis system;

[0107] S2 capillary column pretreatment: Use uncoated quartz capillaries with an inner diameter of 25 μm and a total length of 87 cm; before first use, rinse sequentially: 1 M NaOH solution for 30 minutes, deionized water for 5 minutes, and running buffer for 10-15 minutes; repeat the following steps before each injection: rinse with deionized water for 2 minutes and rinse with running buffer for 2 minutes.

[0108] S3 electrophoresis analysis conditions: (1) Injection method: pressure injection, injection pressure: 0.3 psi; injection time: 3 seconds; (2) Operating conditions: separation voltage: 15–20 kV, separation current: 0.2 kV. μ A. Capillary temperature: constant at 25°C; total electrophoresis time: approximately 30 minutes; detection system: used to detect FITC-derived amino acids, fluorescence detector, excitation wavelength 485 nm, emission wavelength 520 nm;

[0109] S4 Sample processing and FITC derivatization: Take 1 mL of serum acetonitrile solution and add 1.2 mL of FITC (fluorescein isothiocyanate) acetonitrile solution (2 mmol / L); react in a 30℃ water bath for 1 hour to complete the fluorescent labeling of amino acids; the derivatized sample can be directly used for capillary electrophoresis or stored at -20℃ for later use.

[0110] S5 Analysis: The derivatized test sample is separated and detected according to the electrophoretic analysis conditions in S3 to obtain the electrophoretic peak diagram of the test sample and extract the peak area value of each peak.

[0111] Experimental results: Due to the above settings, the amino acid peaks overlapped severely and some amino acids could not be distinguished, making it impossible to establish an effective standard curve for calibration, resulting in no effective separation and inability to perform quantitative analysis. Figure 22The graph shows the results without the addition of cyclodextrin as a separating reagent; the components are: 1. Arginine, 2. Lysine, 3. Leucine, 4. Isoleucine, 5. Methionine, 6. Phenylalanine, 7. Tryptophan, 8. Tyrosine, 9. Histidine, 10. Cysteine, 11. Valine, 12. Glutamine, 13. Proline, 14. Asparagine, 15. Threonine, 16. Glycine, 17. Serine, 18. Alanine, 19. Glutamic acid, 20. Aspartic acid.

[0112] Comparative Example 2

[0113] Experimental methods:

[0114] Preparation of S1 basal buffer (running buffer): Sodium tetraborate decahydrate (Borax) 90 mmol / L, sodium taurocholate 2 mmol / L. α -Cyclodextrin 80 mmol / L, β - Cyclodextrin 20 mmol / L, adjust pH to 9.0 (using 1M NaOH or HCl). After the solution is prepared, use 0.22... μ Microporous membrane filtration is used to prevent particles from affecting the stability of the electrophoresis system;

[0115] S2 capillary column pretreatment: using an inner diameter of 25 mm μ Uncoated quartz capillary tube with a total length of 87 cm and a diameter of m; before first use, rinse sequentially: 1 M NaOH solution for 30 minutes, deionized water for 5 minutes, and running buffer for 10-15 minutes; repeat the following steps before each injection: rinse with deionized water for 2 minutes and rinse with running buffer for 2 minutes.

[0116] S3 electrophoresis analysis conditions: (1) Injection method: pressure injection, injection pressure: 0.3 psi; injection time: 3 seconds; (2) Operating conditions: separation voltage: 15–20 kV, separation current: 0.2 μ A. Capillary temperature: constant at 25°C; total electrophoresis time: approximately 30 minutes; detection system: used to detect FITC-derived amino acids, fluorescence detector, excitation wavelength 485 nm, emission wavelength 520 nm;

[0117] S4 Sample processing and FITC derivatization: Take 1 mL of serum acetonitrile solution and add 1.2 mL of FITC (fluorescein isothiocyanate) acetonitrile solution (2 mmol / L); react in a 30℃ water bath for 1 hour to complete the fluorescent labeling of amino acids; the derivatized sample can be directly used for capillary electrophoresis or stored at -20℃ for later use.

[0118] S5 Analysis: The sample to be tested is separated and detected by a capillary electrophoresis system to obtain the electrophoretic peak diagram of each target amino acid and extract the peak area value of each peak.

[0119] Experimental results: Due to the above settings, the amino acid peaks overlapped severely and some amino acids could not be distinguished, making it impossible to establish an effective standard curve for calibration, resulting in no effective separation and inability to perform quantitative analysis. Figure 23 Add only α -、 β - Cyclodextrins cannot separate 20 amino acids, with some overlap. Histidine / cysteine / valine (9 / 10 / 11) and glycine / serine (16 / 17) overlap, making quantification of these amino acids impossible. Specifically: 1. Arginine; 2. Lysine; 3. Leucine; 4. Isoleucine; 5. Methionine; 6. Phenylalanine; 7. Tryptophan; 8. Tyrosine; 9. Histidine; 10. Cysteine; 11. Valine; 12. Glutamine; 13. Proline; 14. Asparagine; 15. Threonine; 16. Glycine; 17. Serine; 18. Alanine; 19. Glutamic acid; 20. Aspartic acid.

[0120] Comparative Example 3

[0121] Experimental methods:

[0122] Preparation of S1 basal buffer (running buffer): Sodium tetraborate decahydrate (Borax) 90 mmol / L, sodium taurocholate 2 mmol / L. α -Cyclodextrin 80 mmol / L, β -Cyclodextrin 30 mmol / L, γ- Add 20 mmol / L cyclodextrin and adjust the pH to 9.0 (using 1M NaOH or HCl). After the solution is prepared, filter it through a 0.22 μm microporous membrane to avoid particles affecting the stability of the electrophoresis system.

[0123] S2 capillary column pretreatment: using an inner diameter of 25 mm μ Uncoated quartz capillary tube with a total length of 87 cm and a diameter of m; before first use, rinse in the following order: 1 M NaOH solution for 30 minutes, deionized water for 5 minutes, and running buffer for 10-15 minutes; repeat the following steps before each injection: rinse with deionized water for 2 minutes and rinse with running buffer for 2 minutes.

[0124] S3 electrophoresis analysis conditions: (1) Injection method: pressure injection, injection pressure: 0.3 psi; injection time: 3 seconds; (2) Operating conditions: separation voltage: 15~20 kV, separation current: 0.2 kV. μA. Capillary temperature: constant at 25°C; total electrophoresis time: approximately 30 minutes; detection system: used to detect FITC-derived amino acids, fluorescence detector, excitation wavelength 485 nm, emission wavelength 520 nm;

[0125] S4 Sample processing and FITC derivatization: Take 1 mL of serum acetonitrile solution and add 1.2 mL of FITC (fluorescein isothiocyanate) acetonitrile solution (2 mmol / L); react in a 30℃ water bath for 1 hour to complete the fluorescent labeling of amino acids; the derivatized sample can be directly used for capillary electrophoresis or stored at -20℃ for later use.

[0126] S6 Analysis: The sample to be tested is separated and detected by a capillary electrophoresis system to obtain the electrophoretic peak diagram of each target amino acid and extract the peak area value of each peak.

[0127] Experimental results: Due to the above settings, amino acid peaks severely overlapped, and some amino acids were indistinguishable, making it impossible to establish an effective standard curve for calibration. Effective separation was not achieved, and quantitative analysis was impossible. See also... Figure 24 Using cyclodextrin in a different proportion than the invented reagent as the separating reagent ( α -、 β -、 γ - When three cyclodextrins were used in a fixed ratio (80:30:20 mmol / L), 20 amino acids could not be separated. There was overlap between proline / asparagine (13 / 14) and glycine / serine (16 / 17), making quantitative analysis impossible. The amino acids were: 1. Arginine; 2. Lysine; 3. Leucine; 4. Isoleucine; 5. Methionine; 6. Phenylalanine; 7. Tryptophan; 8. Tyrosine; 9. Histidine; 10. Cysteine; 11. Valine; 12. Glutamine; 13. Proline; 14. Asparagine; 15. Threonine; 16. Glycine; 17. Serine; 18. Alanine; 19. Glutamic acid; 20. Aspartic acid.

[0128] (III) Methodological Examination

[0129] To verify the reliability and applicability of the method of the present invention, the capillary electrophoresis serum amino acid analysis method was subjected to the following methodological investigation:

[0130] 3.1 Examine repeatability and precision

[0131] The same sample solution was selected and injected six times consecutively under the same conditions. The migration time and peak area of ​​each amino acid were then measured. The results showed that the relative standard deviation (RSD) of the migration time of each amino acid was less than 2.0%, and the RSD of the peak area was less than 3.0%, indicating that the method has good repeatability and precision.

[0132] 3.2 Linear range and detection limit

[0133] Using the standard curve established in Example S4, the results showed that all 20 amino acids exhibited good linearity in the concentration range of 20–500 μM, with correlation coefficients (R²) greater than 0.995. The limits of detection (LOD) calculated based on the signal-to-noise ratio (S / N=3) were 1–5. μ M, with a limit of quantitation (LOQ) calculated using S / N=10, is 5~10. μ M can meet the needs of amino acid detection in clinical serum samples.

[0134] 3.3 Spike Recovery Rate

[0135] Amino acid standard solutions of known concentrations were added to a blank serum matrix, processed and detected using the method of this invention, and the recoveries were calculated. The results showed that the recoveries of the 20 amino acids were between 92% and 108%, with RSDs less than 5%, indicating that this method has good adaptability to complex matrix samples.

[0136] 3.4 Stability Test

[0137] The derivatized samples were placed at room temperature and -20℃ for different periods of time and then tested. The results showed that the signal of the sample did not significantly decrease within 12 hours at room temperature, and it was stable for more than 7 days when stored at -20℃, indicating that the sample derivatization process of this method has good stability.

Claims

1. A method for capillary electrophoresis analysis of serum amino acids, characterized by, Comprising the following steps: S1 running buffer preparation: running aqueous buffer containing sodium tetraborate decahydrate 80-100 mmol / L, sodium taurocholate 1-3 mmol / L, α- cyclodextrin 70-90 mmol / L, β- cyclodextrin 10-30 mmol / L, Gamma cyclodextrin 5-20 mmol / L, adjust pH to 9.0, filter for use; S2 quartz capillary column selection and pretreatment: select uncoated quartz capillary column, use before flushing with NaOH solution, deionized water flushing, running buffer flushing, before each injection, respectively with deionized water and running buffer flushing; S3 electrophoresis analysis conditions: (1) injection mode: using pressure injection mode; (2) running conditions: set the separation voltage, separation current, capillary temperature, electrophoresis time, respectively, (3) detector: fluorescence detector; S4: Draw the quasi-curve: by configuring 5 groups of different concentrations of standard amino acid solution, the concentrations are 20 Mu M, 50 Mu M, 100 Mu M, 250 Mu M and 500 Mu M, sequentially detect under the electrophoresis analysis conditions described in S3, record the corresponding electrophoresis peak area of each standard sample, take the amino acid concentration as the abscissa and the peak area as the ordinate, draw the standard curve, and establish the linear or nonlinear function relationship between the amino acid concentration and the peak area; S5 sample derivatization: using the concentration of 1~3mmol / L of fluorescein isothiocyanate acetonitrile solution derivatization of the sample to be measured, the solution solvent and the sample to be measured is consistent; S6 quantitative analysis: the derivatization of the sample to be measured according to S3 electrophoresis analysis conditions for separation and detection, obtain the electrophoresis peak chart of the sample to be measured, extract the peak area value of each peak, the peak area obtained into the function relationship in step S4 is constructed, calculate the concentration of amino acids in the sample to be measured.

2. The method for capillary electrophoresis analysis of serum amino acids according to claim 1, characterized by, In S1, the filtration employs 0.2-0.6 Mu m microneedle filter.

3. The method for capillary electrophoresis analysis of serum amino acids according to claim 1, characterized by, In S2, the concentration of NaOH solution is 0.5~2M, the flushing time is 20~60min.

4. The method for capillary electrophoresis analysis of serum amino acids according to claim 1, characterized by, In S3, the pressure injection mode is used, the injection pressure is 0.1~0.5 psi, and the injection time is 1~5s.

5. The method for capillary electrophoresis analysis of serum amino acids according to claim 1, characterized by, In S3, the running condition is respectively set as separation voltage 15~20 kV, separation current 0.1~0.3 Mu A, capillary temperature 20~30℃, electrophoresis time 20~60 min.

6. The method for capillary electrophoresis analysis of serum amino acids according to claim 1, characterized by, In S3, the excitation wavelength of the fluorescence detector is 485 nm, and the emission wavelength is 520 nm.

7. The method for capillary electrophoresis analysis of serum amino acids according to claim 1, characterized by, In S4, the standard amino acid solution is a mixed standard solution prepared from 20 kinds of amino acid standard samples.

8. The method for capillary electrophoresis analysis of serum amino acids according to claim 1, characterized by, In S4, the function relationship between the amino acid concentration and the peak area is: Leucine y = 1690x, R 2 = 0.999; Tryptophan y = 519.2x - 4196, R 2 = 0.997; Isoleucine y = 1828x - 1906, R2= 0.998; 2 = 0.998; Phenylalanine y = 2513.3x - 23845, R 2 = 0.9983; Methionine y = 3144x - 20346, R 2 = 0.9937; Valine y = 1026.1x + 5227.4, R 2 = 0.9969; Aspartic acid y = 2214.1x - 5664.8, R = 0.9964; 2 = 0.9964; Proline y = 2077.9x + 30996, R 2 = 0.9953; Lysine y = 126.62x - 729.31, R 2 = 0.9952; Serine y = 261.76x - 2742.2, R 2 = 0.999; Threonine y = 81.319x + 886.3, R 2 = 0.9978; Histidine y = 310.15x - 5629.9, R 2 = 0.9988; Tyrosine y = 500.42x - 136.57, R 2 = 0.9976; Alanine y = 172.08x - 3774.2, R 2 = 0.9972; Glutamine y = 103.91x + 5815.3, R 2 = 0.9981; Glycine y = 322.73x + 3896.9, R 2 = 0.9975; Glutamic acid y = 378.93x - 4742.8, R 2 = 0.9978; Asparagine y = 51.599x - 892.7, R 2 = 0.9991; Cysteine y = 318.38x - 77.129, R 2 = 0.998; Arginine y = 384.47x + 7.6703, R 2 = 0.9984.

9. The method for capillary electrophoresis analysis of serum amino acids according to claim 1, characterized by, In S5, the step of sample derivatization comprises: taking the sample to be tested and adding 1-3 mmol / L of fluorescein isothiocyanate acetonitrile solution with a volume of 1.2 times that of the sample, and reacting at 20-40°C for 40 min-2h to complete the fluorescence labeling of the amino acid, and then using 0.2-0.6 Mu m micropore needle filter, direct injection or -30 to -10°C storage for standby.

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