A method for determining methionine and its impurities by high performance liquid chromatography

Abstract

The application provides a detection method for determining methionine and impurities thereof by using high performance liquid chromatography, comprising the following steps: A) dissolving a sample to be detected by using water to obtain a test sample solution; B) determining the test sample solution by using high performance liquid chromatography to obtain a chromatogram of the test sample solution; and chromatographic parameters are as follows: a chromatographic column is an XB-SCX column; a mobile phase is an isocratic elution; and the mobile phase is a water solution containing 0.09%-0.11% formic acid and 0.09%-0.11% diethylamine. The application determines methionine and impurities thereof by using HPLC analysis and detection spectrum, and other components in the test sample solution do not interfere with the detection of methionine sulfoxide and serine; the application can achieve the purpose of rapidly, effectively and simultaneously detecting methionine sulfoxide and serine in methionine, and has good specificity, durability and high detection sensitivity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for determining methionine and its impurities using high performance liquid chromatography. Background Technology

[0002] Methionine, also known as methionine, is used in the Chinese Pharmacopoeia for the detection of other amino acids (serine) via thin-layer chromatography. This method is cumbersome and, in particular, has low sensitivity. Methionine is easily oxidized and degraded to produce methionine sulfoxide impurities, which in turn oxidize to methionine sulfones.

[0003] Current technologies cannot simultaneously detect methionine sulfoxide and serine in methionine. Therefore, a method for determining methionine and its impurities using high-performance liquid chromatography is essential. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for determining methionine and its impurities using high performance liquid chromatography. The method of the present invention has high separation, low detection limit, high sensitivity, and accurate detection results.

[0005] This invention provides a method for determining methionine and its impurities using high-performance liquid chromatography, comprising:

[0006] A) Dissolve the sample to be tested in water to obtain the test solution;

[0007] B) The test solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the test solution;

[0008] The chromatographic parameters are:

[0009] The chromatographic column was an XB-SCX column; isocratic elution was used; the mobile phase was an aqueous solution containing 0.09%–0.11% formic acid and 0.09%–0.11% diethylamine.

[0010] Preferably, the method further includes preparing a reference solution: dissolving methionine, serine, and methionine sulfoxide in water to obtain a reference solution;

[0011] The reference solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the test solution were qualitatively or quantitatively determined based on the chromatogram of the reference.

[0012] Preferably, the mobile phase is an aqueous solution containing 0.1% formic acid and 0.09% diethylamine.

[0013] Preferably, the chromatographic column is an Ultimate XB-SCX column with dimensions of 250×4.6mm and 5μm.

[0014] Preferably, the column temperature is 30°C.

[0015] Preferably, the injection volume is 20 μL and the flow rate is 1.0 ml / min.

[0016] Preferably, the detection wavelength is 210 nm.

[0017] Preferably, the impurities include serine and methionine sulfoxide;

[0018] The quantitative detection concentration of methionine sulfoxide is 0.32 μg / ml, with a limit of 0.006% and a minimum detection concentration of 0.16 μg / ml, with a limit of 0.0032%; the quantitative detection concentration of serine is 3.2 μg / ml, with a limit of 0.065% and a minimum detection concentration of 1.6 μg / ml, with a limit of 0.032%.

[0019] Preferably, the separation degree of methionine sulfoxide and serine is 1.72 to 2.17; and the separation degree of serine and methionine is 1.72 to 1.84.

[0020] Preferably, in the reference solution, the injection concentration of serine solution is 1.62–25.92 μg / mL; and the injection concentration of methionine sulfoxide is 0.16–25.25 μg / mL.

[0021] Compared with existing technologies, this invention provides a method for determining methionine and its impurities using high-performance liquid chromatography (HPLC), comprising: A) dissolving the sample to be tested in water to obtain a test solution; B) analyzing the test solution using HPLC to obtain a chromatogram of the test solution; the chromatographic parameters are: an XB-SCX column; isocratic elution of the mobile phase; and an aqueous solution containing 0.09%–0.11% formic acid and 0.09%–0.11% diethylamine. This invention uses HPLC analysis to determine methionine and its impurities, and other components in the test solution do not interfere with the detection of methionine sulfoxide and serine. This invention can achieve rapid and effective simultaneous detection of methionine sulfoxide and serine in methionine, exhibiting good specificity, robustness, and high detection sensitivity. Attached Figure Description

[0022] Figure 1 This is a chromatogram of the system suitability solution in the methodological investigation of Example 5;

[0023] Figure 2 This is a chromatogram of the system suitability solution from Example 1;

[0024] Figure 3 The chromatogram of the system suitability solution for Example 2 is shown.

[0025] Figure 4 The chromatogram of the system suitability solution for Example 3 is shown.

[0026] Figure 5 This is a chromatogram of the system suitability solution for mobile phase 3 in Example 4;

[0027] Figure 6 This is a chromatogram of the system suitability solution for mobile phase 4 in Example 4;

[0028] Figure 7 Linear relationship diagram of serine;

[0029] Figure 8 Linear relationship of methionine sulfoxide;

[0030] Figure 9 The chromatogram is for Comparative Example 1;

[0031] Figure 10 This is the chromatogram of Comparative Example 2. Detailed Implementation

[0032] This invention provides a method for determining methionine and its impurities using high-performance liquid chromatography (HPLC). Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0033] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0034] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0035] This invention addresses the limitation of existing analytical methods in simultaneously determining methionine sulfoxide and serine, related substances of methionine, using high-performance liquid chromatography (HPLC). This method is convenient, rapid, requires no derivatization, and has low detection costs. The quantitative detection concentration of methionine sulfoxide is 0.32 μg / ml, with a minimum detection concentration of 0.16 μg / ml; the quantitative detection concentration of serine is 3.2 μg / ml, with a minimum detection concentration of 1.6 μg / ml. Experiments show that other components in the sample solution do not interfere with the detection of methionine sulfoxide and serine. This invention enables rapid and effective simultaneous detection of methionine sulfoxide and serine in methionine, exhibiting good specificity, robustness, and high detection sensitivity, filling a gap in existing technologies.

[0036] This invention provides a method for determining methionine and its impurities using high-performance liquid chromatography, comprising:

[0037] A) Dissolve the sample to be tested in water to obtain the test solution;

[0038] B) The test solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the test solution;

[0039] The chromatographic parameters are:

[0040] The chromatographic column is an XB-SCX column; the mobile phase is isocratic elution; the mobile phase is an aqueous solution containing 0.09%–0.11% formic acid and 0.09%–0.11% diethylamine.

[0041] The samples to be tested in this invention include products containing methionine and its impurities.

[0042] Methionine sulfoxide is obtained by the oxidation and destruction of methionine.

[0043] Sample information:

[0044] Methionine 20230207 Xiuzheng Pharmaceutical Group Liuhe Pharmaceutical Co., Ltd. Methionine 230401 Tonghua Youkang Pharmaceutical Co., Ltd. Serine P2075348 Shanghai Titan Technology Co., Ltd.

[0045] The impurities described in this invention include methionine sulfoxide and serine.

[0046] The method for determining methionine and its impurities by high performance liquid chromatography provided by the present invention first involves dissolving the sample to be tested in water to obtain a test solution.

[0047] The sample to be tested is dissolved in water to obtain the test solution.

[0048] The present invention utilizes the above-mentioned extraction solvent, which provides a large amount of chromatographic peak information and yields good results.

[0049] In one specific embodiment of the present invention, the mass-to-volume ratio of the sample to be tested and water is 5 mg: 1 mL.

[0050] The present invention also includes the preparation of a reference solution: methionine, serine and methionine sulfoxide are dissolved in water respectively to obtain a reference solution.

[0051] According to the present invention, in the reference solution, the injection concentration of serine solution is 1.62–25.92 μg / mL; and the injection concentration of methionine sulfoxide is 0.16–25.25 μg / mL.

[0052] The test solution was analyzed by high performance liquid chromatography (HPLC) to obtain a chromatogram of the test solution; the reference solution was analyzed by HPLC to obtain a chromatogram of the reference; and the components of the chromatogram of the test solution were qualitatively or quantitatively determined based on the chromatogram of the reference.

[0053] The chromatographic parameters are:

[0054] The chromatographic column was an XB-SCX column;

[0055] The XB-SCX column of this invention is a strong cation exchange column, and the packing material is a silica matrix with phenylsulfonic acid groups bonded on it.

[0056] In one embodiment of the present invention, the chromatographic column is an Ultimate XB-SCX chromatographic column with dimensions of 250×4.6mm and 5μm.

[0057] The present invention achieves symmetrical chromatographic peaks and good resolution under the above-mentioned chromatographic column conditions.

[0058] According to the present invention, the above is isocratic elution.

[0059] In a preferred embodiment of the present invention, the mobile phase is an aqueous solution containing 0.09% to 0.11% formic acid and 0.09% to 0.11% diethylamine.

[0060] In this invention, the peaks of methionine, methionine sulfoxide, and serine are well separated and do not interfere with each other, and the response values ​​and peak shapes are also good.

[0061] In one preferred embodiment of the present invention, the mobile phase is an aqueous solution containing 0.1% formic acid and 0.09% diethylamine.

[0062] The present invention creatively discovers that when the mobile phase composition is mobile phase 3: water (containing 0.1% formic acid and 0.09% diethylamine), the separation between peaks is better than that of other mobile phases.

[0063] In some preferred embodiments, the column temperature is 30°C.

[0064] The chromatographic column of this invention exhibits symmetrical peaks and good resolution under the above-mentioned 30℃ conditions.

[0065] In some preferred embodiments, the injection volume is 20 μL;

[0066] In some preferred embodiments, the flow rate is 1.0 ml / min.

[0067] The present invention found that the chromatographic peaks were well separated and the peak shapes were more symmetrical at a flow rate of 1 ml / min, which is the optimal solution.

[0068] In some preferred embodiments, the detection wavelength is 210 nm.

[0069] The inventors have discovered that the chromatographic information is abundant at 210 nm, all components have good absorption, the response value is moderate, the peaks are well separated, and the baseline is stable.

[0070] The quantitative detection concentration of methionine sulfoxide described in this invention is 0.32 μg / ml, with a limit of 0.006% and a minimum detection concentration of 0.16 μg / ml, with a limit of 0.0032%; the quantitative detection concentration of serine is 3.2 μg / ml, with a limit of 0.065% and a minimum detection concentration of 1.6 μg / ml, with a limit of 0.032%. The resolution between methionine sulfoxide and serine described in this invention is 1.72–2.17; the resolution between serine and methionine is 1.72–1.84.

[0071] As can be seen from the above, the methionine sulfoxide, serine and methionine of the present invention have high separation degree and strong specificity.

[0072] This invention provides a method for determining methionine and its impurities using high-performance liquid chromatography (HPLC), comprising: A) dissolving the sample in water to obtain a test solution; B) analyzing the test solution using HPLC to obtain a chromatogram of the test solution; the chromatographic parameters are: an XB-SCX column; isocratic elution of the mobile phase; and an aqueous solution containing 0.09%–0.11% formic acid and 0.09%–0.11% diethylamine. This invention uses HPLC analysis to determine methionine and its impurities, and other components in the test solution do not interfere with the detection of methionine sulfoxide and serine. This invention achieves rapid and effective simultaneous detection of methionine sulfoxide and serine in methionine, exhibiting good specificity, robustness, and high detection sensitivity.

[0073] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for determining methionine and its impurities using high-performance liquid chromatography.

[0074] Example 1

[0075] High performance liquid chromatography was used with an Ultimate XB-SCX column, 250×4.6mm, 5μm.

[0076] Chromatographic conditions:

[0077] Flow rate: 1.0 ml / min;

[0078] Column temperature: 30℃;

[0079] Wavelength: 210nm

[0080] Injection volume: 20 μL;

[0081] Mobile phase: Water (containing 0.1% formic acid and 0.1% diethylamine)

[0082] Solvent: Water

[0083] Test solution: Accurately weigh an appropriate amount of methionine, dissolve it in water and quantitatively dilute it to a solution containing 5 mg of methionine per 1 ml.

[0084] Degradation of the test solution (methionine sulfoxide solution): Accurately weigh 20 mg of methionine, dissolve it in 5 ml of water, add 0.5 ml of 30% hydrogen peroxide, let stand for 30 min, then dilute to 20 ml with water and shake well to obtain the methionine sulfoxide stock solution; then accurately measure 0.25 ml of the stock solution, place it in a 10 ml volumetric flask, dilute with water and dilute to the mark, shake well to obtain the solution (25 μg / ml).

[0085] Serine localization solution: Accurately weigh an appropriate amount of serine, dissolve it in water and quantitatively dilute it to a solution containing 25 μg of serine per 1 ml.

[0086] System suitability solution: Take an appropriate amount of sample, accurately weigh it, and accurately measure an appropriate amount of serine and methionine sulfoxide stock solution. Dissolve and dilute them with diluent to prepare a mixed solution containing approximately 5 mg of methionine and approximately 25 μg each of serine and methionine sulfoxide impurities.

[0087] Figure 2 The chromatogram for Example 1 is shown below. Discussion: In this method, the retention times of methionine, methionine sulfoxide, and serine are 4.9 min, 4.0 min, and 4.5 min, respectively. The peaks are well separated and do not interfere with each other, and the response values ​​and peak shapes are good.

[0088] Example 2

[0089] Adjust the amounts of formic acid and diethylamine in the mobile phase. Mobile phase 1: water (containing 0.09% formic acid and 0.1% diethylamine), the rest is the same as in Example 1. Figure 3 This is the chromatogram of Example 2.

[0090] Example 3

[0091] Adjust the amounts of formic acid and diethylamine in the mobile phase. Mobile phase 2: water (containing 0.11% formic acid and 0.1% diethylamine), the rest is the same as in Example 1. Figure 4 This is the chromatogram of Example 3.

[0092] Example 4

[0093] Adjust the amounts of formic acid and diethylamine in the mobile phase: mobile phase 3: water (containing 0.1% formic acid and 0.09% diethylamine), mobile phase 4: water (containing 0.1% formic acid and 0.11% diethylamine).

[0094] Figure 5 and Figure 6 The chromatogram for Example 4 shows that the inventors found that the resolution between peaks in this invention is better than that of the mobile phase in other examples.

[0095] Comparative Example 1

[0096] High performance liquid chromatography was used with an Ultimate AQ-C18 column, 250×4.6mm, 5μm.

[0097] Chromatographic conditions:

[0098] Flow rate: 1.0 ml / min;

[0099] Column temperature: 30℃;

[0100] Wavelength: 210nm

[0101] Injection volume: 20 μl;

[0102] Mobile phase: Water (containing 0.1% formic acid and 0.1% diethylamine)

[0103] Solvent: Water

[0104] Test solution: Accurately weigh an appropriate amount of methionine, dissolve it in water and dilute it quantitatively to a solution containing 1 mg of methionine per 1 ml.

[0105] Destroy the test solution (methionine sulfoxide solution): Accurately weigh 20 mg of methionine, dissolve it in 5 ml of water, add 0.5 ml of 30% hydrogen peroxide, let it stand for 30 min, then dilute to 20 ml with water and shake well to obtain the solution.

[0106] Figure 9 The chromatogram is for Comparative Example 1. Discussion: In this method, the blank solution interferes with the elution of methionine peak, and methionine sulfoxide does not elute.

[0107] Comparative Example 2

[0108] High performance liquid chromatography was used with an Ultimate XB-SCX column, 250×4.6mm, 5μm.

[0109] Chromatographic conditions:

[0110] Flow rate: 1.0 ml / min;

[0111] Column temperature: 30℃;

[0112] Wavelength: 210nm

[0113] Injection volume: 20 μL;

[0114] Mobile phase: water (containing 0.2% formic acid and 0.1% diethylamine)

[0115] Solvent: Water

[0116] Test solution: Accurately weigh an appropriate amount of methionine, dissolve it in water and dilute it quantitatively to a solution containing 1 mg of methionine per 1 ml.

[0117] Destroy the test solution (methionine sulfoxide solution): Accurately weigh 20 mg of methionine, dissolve it in 5 ml of water, add 0.5 ml of 30% hydrogen peroxide, let it stand for 30 min, then dilute to 20 ml with water and shake well to obtain the solution.

[0118] Serine localization solution: Accurately weigh an appropriate amount of serine, dissolve it in water and quantitatively dilute it to a solution containing 1 mg of serine per 1 ml.

[0119] Figure 10 The chromatogram is for Comparative Example 2. Discussion: In this method, the retention times of methionine, methionine sulfoxide, and serine are 6.408 min, 4.844 min, and 5.586 min, respectively. The peaks are well separated and do not interfere with each other. However, the response value of serine is small, and the peak shape needs to be optimized.

[0120] Example 5 Methodological Investigation

[0121] Diluent: Filtered purified water

[0122] Blank solution: diluent

[0123] Serine stock solution: Accurately weigh an appropriate amount of serine reference standard, dissolve it with diluent and quantitatively dilute it to prepare a solution containing approximately 0.1 mg per 1 ml.

[0124] Serine positioning solution: Accurately measure an appropriate amount of serine stock solution, dissolve it with diluent and quantitatively dilute it to prepare a solution containing approximately 25 μg per 1 ml.

[0125] Methionine sulfoxide stock solution: Accurately weigh approximately 20 mg of methionine, dissolve it in 5 ml of diluent, add 0.5 ml of 30% hydrogen peroxide, let stand at room temperature for 30 min, dilute with diluent and bring the volume to 20 ml, mix well, and use for later use. (1 mg / ml)

[0126] Methionine sulfoxide positioning solution: Accurately measure an appropriate amount of methionine sulfoxide stock solution, dissolve it with diluent and quantitatively dilute it to prepare a solution containing approximately 25 μg per 1 ml.

[0127] Test solution: Take an appropriate amount of sample, weigh it accurately, dissolve it with diluent and dilute it quantitatively to prepare a solution containing about 5 mg per 1 ml.

[0128] Self-control solution: Accurately measure an appropriate amount of the test solution and dilute it with diluent to prepare a solution containing approximately 25 μg per 1 ml.

[0129] System suitability solution: Accurately weigh an appropriate amount of sample, and accurately measure appropriate amounts of serine and methionine sulfoxide stock solutions. Dissolve and dilute with diluent to prepare a mixed solution containing approximately 5 mg of methionine and approximately 25 μg each of serine and methionine sulfoxide impurities. (For example: accurately weigh approximately 50 mg of sample, accurately measure 2.5 ml of serine stock solution and 0.25 ml of methionine sulfoxide stock solution, place them together in a 10 ml volumetric flask, dissolve with diluent and bring to the mark, mix well, and the solution is ready.)

[0130] The test was conducted using the limit method, with a limit of 0.5%, and the results are as follows:

[0131] (1) System adaptability and specificity:

[0132] Blank solution, system suitability solution, self-control solution (6 consecutive injections), test solution, and various positioning solutions were prepared. Results and chromatograms are as follows:

[0133] Table 1 System Applicability and Specificity Results

[0134]

[0135] Table 2. Repeatability results of the self-control solution

[0136]

[0137]

[0138] Figure 1 This is the chromatogram for the system suitability solution. In the system suitability solution, the resolution between each peak is greater than 1.5, and the theoretical plate number of methionine sulfoxide and serine is greater than 3000, indicating good specificity.

[0139] (2) Instrument precision

[0140] Table 3

[0141]

[0142] The self-control solution was injected six times consecutively, with a peak area RSD of 0.1% and a theoretical plate number greater than 5000, indicating good precision.

[0143] (3) Solution stability

[0144] Blank solution and test solution were injected at 0, 2, 4, 6, 8, 12, 16, 24 hours and 3 days, respectively.

[0145] Table 4 Solution stability results

[0146]

[0147] For the self-control solution, the changes in peak area at each time point within 3 days compared to 0 hour were all less than 2.0%, and the changes in methionine sulfoxide impurity content detected in the test solution were all less than 10.0%. Both the self-control solution and the test solution were stable at room temperature for 3 days.

[0148] (4) Durability

[0149] Inject blank solution, system suitability solution and test solution under different chromatographic conditions.

[0150] Table 5. Durability Results (System Suitability Solution)

[0151]

[0152] Table 6. Robustness Results (Test Solution)

[0153]

[0154] Conclusion: When a single variable in the chromatographic conditions is slightly varied, the ranges of variation for each condition are as follows: flow rate 1.0 ± 0.1 ml / min, column temperature 30 ± 2℃, wavelength 210 ± 2 nm, mobile phase (water containing 0.1% ± 0.01% formic acid and 0.1% ± 0.01% diethylamine). Under these chromatographic conditions, the resolution between each peak in the system suitability solution is greater than 1.5, and the theoretical plate numbers for methionine sulfoxide and serine are both greater than 3000. The variation in the content of methionine sulfoxide impurities detected in the test solution is less than 20.0%. The system exhibits good robustness.

[0155] (5) Results of limit of quantitation and limit of detection:

[0156] The serine localization solution and methionine sulfoxide localization solution were serially diluted to determine the limit of quantitation and the limit of detection. The limit of quantitation solution was injected continuously for 6 injections, and the limit of detection solution was injected continuously for 3 injections.

[0157] Table 7 Results of Limit of Quantitation and Limit of Detection

[0158]

[0159] Table 8. Repeatability results of limit of quantitation and limit of detection

[0160]

[0161] Conclusion: The quantitative detection concentration of methionine sulfoxide was 0.32 μg / ml, with a limit of 0.006% and a minimum detection concentration of 0.16 μg / ml, with a limit of 0.0032%. The quantitative detection concentration of serine was 3.2 μg / ml, with a limit of 0.065% and a minimum detection concentration of 1.6 μg / ml, with a limit of 0.032%. Figure 7 Linear relationship diagram of serine; Figure 8 The graph shows the linear relationship between methionine sulfoxide and its concentration. It can be seen that the concentration range of serine in this invention is 1.62–25.92 μg / ml, and the concentration range of methionine sulfoxide is 0.16–25.25 μg / ml. Specific data are shown in Table 9.

[0162] Table 9

[0163]

[0164]

[0165] 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 method for determining methionine and its impurities using high-performance liquid chromatography, characterized in that, include: A) Dissolve the sample to be tested in water to obtain a test solution; the sample to be tested is manufactured by Tonghua Youkang Pharmaceutical Co., Ltd., batch number 230401 or Xiuzheng Pharmaceutical Group Liuhe Pharmaceutical Co., Ltd., batch number 20230207. B) The test solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the test solution; the detection wavelength was 210 nm. The chromatographic parameters are: The chromatographic column was an XB-SCX column; isocratic elution was used; the mobile phase was an aqueous solution containing 0.09%~0.11% formic acid and 0.09%~0.11% diethylamine. The impurities include serine and methionine sulfoxide.

2. The detection method according to claim 1, characterized in that, It also includes the preparation of a reference solution: methionine, serine and methionine sulfoxide are dissolved in water to obtain a reference solution; The reference solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of the test solution were qualitatively or quantitatively determined based on the chromatogram of the reference.

3. The detection method according to claim 1, characterized in that, The mobile phase is an aqueous solution containing 0.1% formic acid and 0.09% diethylamine.

4. The detection method according to claim 1, characterized in that, The chromatographic column is an Ultimate XB-SCX column with dimensions of 250×4.6mm and 5μm.

5. The detection method according to claim 1, characterized in that, The column temperature was 30℃.

6. The detection method according to claim 1, characterized in that, The injection volume was 20 μL; the flow rate was 1.0 ml / min.

7. The detection method according to claim 1, characterized in that, The quantitative detection concentration of methionine sulfoxide is 0.32 μg / ml, with a limit of 0.006% and a minimum detection concentration of 0.16 μg / ml, with a limit of 0.0032%; the quantitative detection concentration of serine is 3.2 μg / ml, with a limit of 0.065% and a minimum detection concentration of 1.6 μg / ml, with a limit of 0.032%.

8. The detection method according to claim 1, characterized in that, The resolution between methionine sulfoxide and serine is 1.72~2.17; the resolution between serine and methionine is 1.72~1.

84.

9. The detection method according to claim 2, characterized in that, In the reference solution, the injection concentration of serine solution was 1.62~25.92 μg / mL; the injection concentration of methionine sulfoxide was 0.16~25.25 μg / mL.

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