Peptide diagram analysis method of tilpotide and application of peptide diagram analysis method

Abstract

The invention relates to the technical field of medicine analysis, in particular to a peptide diagram analysis method of tilpotide and application of the peptide diagram analysis method. The peptide diagram analysis method of the tilpotide comprises the following steps: (a) adding a Tris-HCl buffer solution and proteolytic enzyme into a sample solution containing a tilpotide sample and a citric acid buffer solution for enzyme digestion reaction and centrifugation to obtain a supernatant; and (b) carrying out high performance liquid chromatography analysis on the supernate. The peptide diagram analysis method of the tilpotide has the advantages of high efficiency, accuracy, good reproducibility and the like, and provides powerful technical support for structural research and characteristic identification of the tilpotide.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a peptide map analysis method of tepoxetine and an application thereof. Background Art

[0002] Tipoictide is a glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist used to treat diabetes, non-alcoholic steatohepatitis (NASH) and chronic weight management. The molecule consists of a 39-amino acid peptide backbone and a side chain at residue 20. Of the 39 amino acids, 37 are natural amino acids and 2 are non-coding aminoisobutyric acid residues at positions 2 and 13. The theoretical sequence of tipoictide is: H-Tyr 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Ile 17 -Ala 18 -Gln 19 -Lys 20 (AEEA-AEEA-γGlu-Eicosanedioic acid)-Ala 21 -Phe 22 -Val 23 -Gln 24 -Trp 25 -Leu 26 -Ile 27 -Ala 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 33 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 -NH2.

[0003] Peptide mapping is based on the molecular weight and amino acid composition of proteins and polypeptides, using highly specific proteolytic enzymes to act on specific peptide chain sites to break the polypeptides into small fragments, and forming characteristic fingerprints through certain separation and detection methods. Peptide mapping is of great significance for the study of polypeptide structure and identification of characteristics. Currently, the prior art does not disclose a peptide mapping method for telportin.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a peptide map analysis method of telpotide and its application in view of the problems existing in the prior art. The peptide map analysis method of telpotide adopts proteolytic enzyme hydrolysis according to the sequence characteristics of telpotide, and separates the characteristic peaks of telpotide through a reversed phase C18 liquid phase system. The method has the characteristics of high efficiency, accuracy and good reproducibility.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] One aspect of the present invention relates to a peptide map analysis method of tepote, comprising the following steps:

[0008] (a) adding Tris-HCl buffer and proteolytic enzyme to a sample solution containing a tepoxetine sample and a citric acid buffer, performing an enzyme digestion reaction and centrifuging to obtain a supernatant;

[0009] (b) subjecting the supernatant to high performance liquid chromatography analysis.

[0010] The peptide map analysis method of telportopeptide has the advantages of high efficiency, accuracy and good reproducibility.

[0011] Preferably, the concentration of the tepoxetine sample in the sample solution is 1 to 2 mg / mL.

[0012] Preferably, the proteolytic enzyme is added in the form of a proteolytic enzyme solution, and the concentration of the proteolytic enzyme solution is 0.01-0.02 μg / μL.

[0013] Preferably, the concentration of the Tris-HCl buffer is 0.5-2M.

[0014] Preferably, the volume ratio of the sample solution, the Tris-HCl buffer and the proteolytic enzyme solution is 25-35:5:25-35.

[0015] Preferably, the proteolytic enzyme comprises: Asp-N enzyme.

[0016] Preferably, the concentration of the citric acid buffer is 5-20 mM.

[0017] Preferably, the temperature of the enzyme cleavage reaction is 20-37°C.

[0018] Preferably, the enzyme digestion reaction time is 24 to 48 hours.

[0019] Preferably, the centrifugal speed is 12500-13500 rpm.

[0020] Preferably, the centrifugation time is 4 to 6 minutes.

[0021] Preferably, the conditions for the HPLC analysis are as follows:

[0022] Mobile phase A is 0.09% to 0.11% trifluoroacetic acid aqueous solution; mobile phase B is 0.09% to 0.11% trifluoroacetic acid acetonitrile solution; the flow rate is 0.1 to 0.4 mL / min; the column temperature is 25 to 45° C.; the detection wavelength is 214 nm; and the injection volume is 2.5 to 10 μL.

[0023] Preferably, the elution procedure of the HPLC analysis is as follows:

[0024] At 0.000min, mobile phase A was 85% and mobile phase B was 15%; from 2.000min to 10.000min, mobile phase A decreased from 85% to 60%, and mobile phase B increased from 15% to 40%; at 25.000min, mobile phase A decreased to 40%, and mobile phase B increased to 60%; from 25.100min to 27.000min, mobile phase A decreased to 20%, and mobile phase B increased to 80%; from 27.100min to 33.000min, mobile phase A recovered to 85%, and mobile phase B recovered to 15%.

[0025] Another aspect of the present invention also relates to a method for studying the structure of telpotide, including the peptide map analysis method of telpotide.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The peptide map analysis method of telpotide provided by the present invention comprises preparing a telpotide sample solution by a specific sampling method, performing an enzymatic cleavage reaction using a specific proteolytic enzyme under certain conditions, and then performing high performance liquid chromatography analysis using a reversed phase C18 liquid phase system to achieve accurate separation and detection of the telpotide peptide map.

[0028] (2) The peptide map analysis method of telpotide provided by the present invention includes three main steps: sample solution preparation, enzyme cleavage reaction and chromatographic analysis, and defines in detail the operating parameters in each step, such as sample concentration, enzyme concentration, enzyme cleavage reaction conditions, chromatographic column type, mobile phase composition, flow rate, column temperature, detection wavelength and elution gradient, etc. It has the characteristics of high efficiency, accuracy and good reproducibility, and provides strong technical support for the structural research and characteristic identification of telpotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 The liquid phase spectrum of the sample after enzyme digestion provided in the embodiment of the present invention comprises characteristic peak 1 (Y1-S8), characteristic peak 2 (D9-L14), characteristic peak 3 (D15-S39) and missed cleavage peptide segment (D9-S39);

[0031] Figure 2 A liquid phase spectrum for detecting undigested samples provided in an embodiment of the present invention;

[0032] Figure 3 A comparative graph of a sample after enzyme digestion and a sample not digested by enzyme digestion provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0033] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0034] One aspect of the present invention relates to a peptide map analysis method of tepote, comprising the following steps:

[0035] (a) adding Tris-HCl buffer and proteolytic enzyme to a sample solution containing a tepoxetine sample and a citric acid buffer, performing an enzyme digestion reaction and centrifuging to obtain a supernatant;

[0036] (b) subjecting the supernatant to high performance liquid chromatography analysis.

[0037] Peptide mapping is based on the molecular weight and amino acid composition characteristics of proteins and polypeptides, using a highly specific proteolytic enzyme to act on a special peptide chain site to break the polypeptide into small fragments, and forming a characteristic fingerprint map through a certain separation and detection means. Peptide mapping is of great significance to the study of polypeptide structure and characteristic identification, but the prior art does not have a peptide mapping method for tepote. The peptide mapping method for tepote provided by the present invention, according to the sequence characteristics of tepote, tepote is enzymatically cleaved by a specific proteolytic enzyme method, has a high enzymatic cleavage efficiency, and then by high performance liquid chromatography analysis, the enzymatic cleavage peptide segments can be accurately separated, and the peptide mapping analysis of tepote is realized.

[0038] The peptide map analysis method of telpotide is to prepare a telpotide sample solution by a specific sampling method, use a specific proteolytic enzyme to perform an enzymatic cleavage reaction under certain conditions, and then use a reversed-phase C18 liquid phase system to perform high performance liquid chromatography analysis to achieve accurate separation and detection of the telpotide peptide map.

[0039] The peptide map analysis method of telportopeptide includes three main steps: sample solution preparation, enzyme cleavage reaction and chromatographic analysis, and defines in detail the operating parameters in each step, such as sample concentration, enzyme concentration, enzyme cleavage reaction conditions, chromatographic column type, mobile phase composition, flow rate, column temperature, detection wavelength and elution gradient.

[0040] The peptide map analysis method of telpotide is characterized by high efficiency, accuracy and good reproducibility, and provides strong technical support for the structural research and property identification of telpotide.

[0041] The telportopide sample was dissolved in a citric acid buffer. The calcium citrate buffer was more likely to maintain pH, allowing the solution to maintain a stable pH value within a certain range.

[0042] Adding the proteolytic enzyme after adding Tris-HCl buffer plays a key role in stabilizing the enzyme activity in the enzyme catalytic reaction, ensuring that the enzyme maintains its optimal catalytic efficiency within a specific pH range.

[0043] Furthermore, the concentration of the citric acid buffer is 5-20 mM, including but not limited to any point value of 5 mM, 7 mM, 9 mM, 11 mM, 13 mM, 15 mM, 17 mM, 19 mM or 20 mM or a range value between any two of them. Limiting the concentration of the citric acid buffer to a certain range can improve the enzymatic cleavage efficiency of the proteolytic enzyme.

[0044] Furthermore, the concentration of the tepoxetine sample in the sample solution is 1 to 2 mg / mL, including but not limited to any point value of 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.6 mg / mL, 1.8 mg / mL or 2 mg / mL or a range value between any two of them.

[0045] Furthermore, the proteolytic enzyme includes but is not limited to: Asp-N enzyme.

[0046] Asp-N enzyme, also known as intracellular protease or aspartate endopeptidase, is an enzyme with a specific catalytic function. Asp-N enzyme can specifically cleave the N-terminus of aspartic acid (Asp) and cysteic acid (or cysteine, which may vary depending on the specific enzyme source and conditions) residues in proteins and peptides. This specificity makes Asp-N enzyme unique in proteomics analysis and protein hydrolysis.

[0047] Furthermore, the proteolytic enzyme is added in the form of a proteolytic enzyme solution, and the concentration of the proteolytic enzyme solution is 0.01-0.02 μg / μL, including but not limited to any point value of 0.01 μg / μL, 0.012 μg / μL, 0.014 μg / μL, 0.016 μg / μL, 0.018 μg / μL or 0.02 μg / μL or a range value between any two of them.

[0048] Further, the concentration of the Tris-HCl buffer is 0.5-2M, including but not limited to any one of 0.5M, 1M, 1.5M or 2M or a range between any two of them.

[0049] Furthermore, the volume ratio of the sample solution, the Tris-HCl buffer and the proteolytic enzyme solution is 25-35:5:25-35, including but not limited to 25:5:35, 27:5:33, 29:5:31, 31:5:29, 33:5:27 or 35:5:25.

[0050] By adding a sample solution with a certain concentration, Tris-HCl buffer and proteolytic enzyme solution according to the above ratio, the dosage ratio of telpotide and proteolytic enzyme can be accurately determined, which is conducive to the full progress of the enzyme cleavage reaction and can also avoid the use of excessive reagents.

[0051] Further, the temperature of the enzyme cleavage reaction is 20-37° C., including but not limited to any point value of 20° C., 23° C., 25° C., 28° C., 30° C., 33° C., 35° C. or 37° C. or any range value between two thereof. According to the activity of the proteolytic enzyme, the appropriate enzyme cleavage reaction temperature is determined to ensure the smooth progress of the enzyme cleavage reaction.

[0052] Further, the enzyme cleavage reaction time is 24 to 48 hours, including but not limited to any point value of 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours or 48 hours or any range value between two of them. The enzyme cleavage reaction is carried out under certain temperature conditions and for a certain time, so that the proteolytic enzyme and telpotide can fully react and complete the enzyme cleavage reaction of telpotide.

[0053] Furthermore, the enzyme cleavage reaction is carried out under light-proof conditions.

[0054] Furthermore, the centrifugal speed is 12500-13500 rpm, including but not limited to any one of 12500 rpm, 12600 rpm, 12700 rpm, 12800 rpm, 12900 rpm, 13000 rpm, 13100 rpm, 13200 rpm, 13300 rpm, 13400 rpm or 13500 rpm or the range between any two of them.

[0055] Furthermore, the centrifugation time is 4 to 6 minutes, including but not limited to any one of 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes or 6 minutes, or a range between any two of them.

[0056] Centrifugation under the above centrifugation conditions can effectively remove impurities in the reaction solution of the enzyme cleavage reaction and improve the accuracy of high performance liquid chromatography analysis.

[0057] Furthermore, the chromatographic column for the HPLC analysis includes, but is not limited to, ACQUITY UPLC BEHC18 (chromatographic column inner diameter 2.1 mm, length 100 mm, particle size 1.7 μm).

[0058] Furthermore, the conditions of the HPLC analysis are as follows:

[0059] Mobile phase A is 0.09% to 0.11% (for example, it can be but not limited to any one of 0.09%, 0.10% or 0.11% or a range between any two) trifluoroacetic acid (TFA) aqueous solution; mobile phase B is 0.09% to 0.11% (for example, it can be but not limited to any one of 0.09%, 0.10% or 0.11% or a range between any two) TFA acetonitrile solution; the flow rate is 0.1 to 0.4 mL / min (for example, it can be but not limited to 0.1 mL / min, 0.2 mL / min, 0.3 mL / min or 0.4 mL / min, any one of the point values ​​or any range values ​​between the two); column temperature is 25-45°C (for example, it can be, but not limited to, any one of 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, 37°C, 39°C, 41°C, 43°C or 45°C, any one of the point values ​​or any range values ​​between the two); detection wavelength is 214nm; injection volume is 2.5-10μL (for example, it can be, but not limited to, any one of 2.5μL, 4μL, 6μL, 8μL or 10μL, any one of the point values ​​or any range values ​​between the two).

[0060] Furthermore, the elution procedure of the high performance liquid chromatography analysis is as follows:

[0061] At 0.000min, mobile phase A was 85% and mobile phase B was 15%; from 2.000min to 10.000min, mobile phase A decreased from 85% to 60%, and mobile phase B increased from 15% to 40%; at 25.000min, mobile phase A decreased to 40%, and mobile phase B increased to 60%; from 25.100min to 27.000min, mobile phase A decreased to 20%, and mobile phase B increased to 80%; from 27.100min to 33.000min, mobile phase A recovered to 85%, and mobile phase B recovered to 15%.

[0062] According to the structural characteristics of telpotide, the present invention improves and adjusts the conditions and elution procedure of high performance liquid chromatography analysis. The conditions of high performance liquid chromatography analysis are combined with a specific elution procedure to accurately separate and detect the telpotide peptide map.

[0063] Another aspect of the present invention also relates to a method for studying the structure of telpotide, including the peptide map analysis method of telpotide.

[0064] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0065] Example 1

[0066] The peptide map analysis method of tepote provided in this embodiment comprises the following steps:

[0067] (1) Sample solution preparation: Weigh an appropriate amount of telportopeptide sample, add an appropriate amount of 10 mM citric acid buffer, the concentration of the sample solution is 1 mg / mL, vortex mix and set aside;

[0068] (2) Preparation of Asp-N solution: Add 2 μg of Asp-N standard to 200 μL of ultrapure water. The concentration of Asp-N solution is 0.01 μg / μL. Vortex mix and set aside.

[0069] (3) Enzyme digestion reaction: Take 30 μL of 1 mg / mL sample solution, then add 5 μL of 1 M Tris-HCl buffer, then add 30 μL of 0.01 μg / μL Asp-N solution, mix well, and react at 25°C in the dark for 24 h; after the reaction, centrifuge at 13000 rpm for 5 min, and take the supernatant for chromatographic analysis;

[0070] (4) Chromatographic conditions:

[0071] Instrument: Thermo Vanquish core with UV detector;

[0072] Chromatographic column: ACQUITY UPLC BEH C18 (2.1x100 mm, 1.7um); Mobile phase A: 0.1% TFA aqueous solution;

[0073] Mobile phase B: 0.1% TFA acetonitrile solution;

[0074] Flow rate: 0.3ml / min;

[0075] Column temperature: 40°C;

[0076] Injection volume: 5ul;

[0077] Detection wavelength: 214nm;

[0078] The elution gradient is shown in Table 1.

[0079] Table 1

[0080] Time (min) Mobile phase A (%) Mobile phase B (%) 0.000 85 15 2.000 85 15 10.000 60 40 25.000 40 60 25.100 20 80 27.000 20 80 27.100 85 15 33.000 85 15

[0081] The results of HPLC test were as follows Figure 1 And as shown in Table 2.

[0082] Table 2

[0083]

[0084]

[0085] The results of HPLC showed that telpotide produced three main peptides and one missed peptide after cleavage by Asp-N enzyme under specific conditions. The peptide map of telpotide could be analyzed by comparing the retention time of the cleaved peptides on HPLC.

[0086] Example 2

[0087] The peptide map analysis method of tepote provided in this embodiment comprises the following steps:

[0088] (1) Sample solution preparation: Weigh an appropriate amount of telportopeptide sample, add an appropriate amount of 20 mM citric acid buffer, the concentration of the sample solution is 1.5 mg / mL, vortex mix and set aside;

[0089] (2) Preparation of Asp-N solution: Add 3 μg of Asp-N standard to 200 μL of ultrapure water. The concentration of Asp-N solution is 0.015 μg / μL. Vortex mix and set aside.

[0090] (3) Enzyme digestion reaction: Take 25 μL of 1.5 mg / mL sample solution, then add 5 μL of 1 M Tris-HCl buffer, then add 35 μL of 0.015 μg / μL Asp-N solution, mix well, and react at 20°C in the dark for 48 h; after the reaction, centrifuge at 13500 rpm for 4 min, and take the supernatant for chromatographic analysis;

[0091] (4) Chromatographic conditions:

[0092] Instrument: Thermo Vanquish core with UV detector;

[0093] Chromatographic column: ACQUITY UPLC BEH C18 (2.1x100 mm, 1.7um);

[0094] Mobile phase A: 0.1% TFA in water;

[0095] Mobile phase B: 0.1% TFA acetonitrile solution;

[0096] Flow rate: 0.1ml / min;

[0097] Column temperature: 25°C;

[0098] Injection volume: 5ul;

[0099] Detection wavelength: 214nm;

[0100] The elution gradient is shown in Table 1.

[0101] Example 3

[0102] The peptide map analysis method of tepote provided in this embodiment comprises the following steps:

[0103] (1) Sample solution preparation: Weigh an appropriate amount of telportopeptide sample, add an appropriate amount of 5 mM citric acid buffer, the concentration of the sample solution is 2 mg / mL, vortex mix and set aside;

[0104] (2) Preparation of Asp-N solution: 4 μg of Asp-N standard was added to 200 μL of ultrapure water. The concentration of Asp-N solution was 0.02 μg / μL. Vortex-mixed and set aside.

[0105] (3) Enzyme digestion reaction: Take 35 μL of 2 mg / mL sample solution, then add 5 μL of 1 M Tris-HCl buffer, then add 25 μL of 0.02 μg / μL Asp-N solution, mix well, and react at 37°C in the dark for 36 h; after the reaction, centrifuge at 12500 rpm for 6 min, and take the supernatant for chromatographic analysis;

[0106] (4) Chromatographic conditions:

[0107] Instrument: Thermo Vanquish core with UV detector;

[0108] Chromatographic column: ACQUITY UPLC BEH C18 (2.1x100 mm, 1.7um);

[0109] Mobile phase A: 0.1% TFA in water;

[0110] Mobile phase B: 0.1% TFA acetonitrile solution;

[0111] Flow rate: 0.4ml / min;

[0112] Column temperature: 45°C;

[0113] Injection volume: 5ul;

[0114] Detection wavelength: 214nm;

[0115] The elution gradient is shown in Table 1.

[0116] Comparative Example 1

[0117] The sample solution was analyzed by high performance liquid chromatography. Figure 2 shown.

[0118] Comparative Example 2

[0119] The only difference from Example 1 is that ultrapure water is used to prepare the sample solution.

[0120] Comparative Example 3

[0121] The only difference from Example 1 is that ultrapure water is used instead of Tris-HCl buffer during the enzyme cleavage reaction.

[0122] Comparative Example 4

[0123] The only difference from Example 1 is that during the enzyme cleavage reaction, the volume ratio of the sample solution, Tris-HCl buffer and proteolytic enzyme solution is 20:5:40.

[0124] Comparative Example 5

[0125] The only difference from Example 1 is that during chromatographic detection, the flow rate is 0.6 mL / min.

[0126] Comparative Example 6

[0127] The only difference from Example 1 is that during chromatographic detection, the elution procedure is as follows:

[0128] At 0.000min, mobile phase A was 75% and mobile phase B was 25%; from 2.000min to 10.000min, mobile phase A decreased from 75% to 55%, and mobile phase B increased from 25% to 45%; at 25.000min, mobile phase A decreased to 30%, and mobile phase B increased to 70%; from 25.100min to 27.000min, mobile phase A decreased to 15%, and mobile phase B increased to 85%; from 27.100min to 33.000min, mobile phase A recovered to 75%, and mobile phase B recovered to 25%.

[0129] Figure 3 It is a comparative spectrum of Example 1 and Comparative Example 1. By comparing the liquid phase spectra of Example 1 and Comparative Example 1, it can be seen that under the enzyme cleavage reaction conditions of Example 1, the Asp-N enzyme has strong specificity and high enzyme cleavage efficiency, and the high-performance liquid phase method can achieve accurate separation of enzyme-cleaved peptide segments.

[0130] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. A peptide map analysis method for tepote, characterized in that: The following steps are involved: (a) adding Tris-HCl buffer and proteolytic enzyme to a sample solution containing a tepoxetine sample and a citric acid buffer, performing an enzyme digestion reaction and centrifuging to obtain a supernatant; (b) subjecting the supernatant to high performance liquid chromatography analysis.

2. The peptide map analysis method of tepoxetine according to claim 1, characterized in that: Includes at least one of the following technical features: (1) The concentration of the tepoxetine sample in the sample solution is 1 to 2 mg / mL; (2) the proteolytic enzyme is added in the form of a proteolytic enzyme solution, and the concentration of the proteolytic enzyme solution is 0.01 to 0.02 μg / μL; (3) The concentration of the Tris-HCl buffer is 0.5-2M.

3. The peptide map analysis method of tepote according to claim 2, characterized in that: The volume ratio of the sample solution, the Tris-HCl buffer and the proteolytic enzyme solution is 25-35:5:25-35.

4. The peptide map analysis method of tepote according to claim 1, characterized in that: The proteolytic enzyme includes: Asp-N enzyme.

5. The peptide map analysis method of tepoxetine according to claim 1, characterized in that: The concentration of the citric acid buffer is 5-20 mM.

6. The peptide map analysis method of tepoxetine according to claim 1, characterized in that: The temperature of the enzyme digestion reaction is 20-37°C; And / or, the enzyme digestion reaction time is 24 to 48 hours.

7. The peptide map analysis method of tepoxetine according to claim 1, characterized in that: The centrifugal speed is 12500-13500 rpm; And / or, the centrifugation time is 4 to 6 minutes.

8. The peptide map analysis method of telpoxetine according to claim 1, characterized in that: The conditions of the HPLC analysis are as follows: Mobile phase A is 0.09% to 0.11% trifluoroacetic acid aqueous solution; mobile phase B is 0.09% to 0.11% trifluoroacetic acid acetonitrile solution; the flow rate is 0.1 to 0.4 mL / min; the column temperature is 25 to 45° C.; the detection wavelength is 214 nm; and the injection volume is 2.5 to 10 μL.

9. The peptide map analysis method of tepote according to claim 1, characterized in that: The elution procedure of the HPLC analysis is as follows: At 0.000min, mobile phase A was 85% and mobile phase B was 15%; from 2.000min to 10.000min, mobile phase A was reduced from 85% to 60% and mobile phase B was increased from 15% to 40%; at 25.000min, mobile phase A was reduced to 40% and mobile phase B was increased to 60%; From 25.100min to 27.000min, mobile phase A was reduced to 20% and mobile phase B was increased to 80%; During the period from 27.100 min to 33.000 min, mobile phase A recovered to 85%, and mobile phase B recovered to 15%.

10. A method for studying the structure of tepote, characterized in that: A peptide map analysis method comprising the telpotide according to any one of claims 1 to 9.

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