CN116908321A

CN116908321A - Protamine sulfate peptide map detection method

Info

Publication number: CN116908321A
Application number: CN202310741595.5A
Authority: CN
Inventors: 陈宗茜; 景秀; 陈松; 鲍海涛; 张昊宁; 汤传根
Original assignee: Nanjing Hanxin Pharmaceutical Technology Co ltd
Current assignee: Nanjing Hanxin Pharmaceutical Technology Co ltd
Priority date: 2023-06-21
Filing date: 2023-06-21
Publication date: 2023-10-20
Anticipated expiration: 2043-06-21

Abstract

The invention relates to a protamine sulfate peptide map detection method, which comprises the following steps: s1: respectively carrying out enzymolysis on the protamine sulfate bulk drug, the reference preparation and/or the standard substance to obtain characteristic peptide fragments; s2: respectively carrying out high performance liquid chromatography tandem mass spectrometry detection on the characteristic peptide fragments from different sources, comparing the retention time, the peak area percentage and the molecular weight of the characteristic peptide Duan Feng, and evaluating the quality of a protamine sulfate sample to be detected; the high performance liquid chromatography uses a hydrophilic chromatographic column Inertsil Amide, wherein a mobile phase A is an aqueous solution of ammonium acetate or ammonium formate, and a mobile phase B is an acetonitrile solution containing an acidic buffer solution. The invention can rapidly evaluate the quality of the protamine sulfate sample to be tested, has guiding significance for perfecting the quality control of the variety, and provides reference for deep understanding of the material basis of the variety.

Description

Protamine sulfate peptide map detection method

Technical Field

The invention relates to the technical field of biological medicines, in particular to a protamine sulfate peptide map detection method.

Background

Protamine is an alkaline protein that exists primarily in the mature sperm cell nuclei of fish (e.g., salmon, trout, herring, etc.) as a DNA-binding nucleoprotein. Protamine was found in 1870 and research into protamine as an antibacterial agent was actively conducted in 1940 to 1960. Protamine is now becoming increasingly used in the food industry and in the medical field. Protamine sulfate is the only anti-heparin drug in extracorporeal circulation cardiac surgery, can counteract the anticoagulation effect of heparin or synthetic anticoagulant, and can be clinically used as antidote for the anticoagulant. Protamine can be combined with various proteins to form a complex, for example, protamine can be combined with insulin to prevent or delay release of insulin and prolong the hypoglycemic effect. When the hormone or the antibacterial agent is compounded with protamine, the drug effect of the hormone or the antibacterial agent can be prolonged, so that the use amount of the hormone or the antibacterial agent is reduced. When 0.5% of protamine is compounded in the anti-influenza drug, the effective period of antibiotics can be prolonged, and the injection dosage of the drug and the injection times of patients can be reduced. In addition, protamine has the effects of resisting tumor, relieving fatigue, enhancing liver function, stimulating pituitary to release gonadotrophin, etc.

The protamine has small molecular weight, is generally composed of about 30-50 amino acids, is rich in arginine, is alkaline, can be dissolved in water and olefine acid, is not easy to dissolve in organic solvents such as ethanol, acetone and the like, has good stability, and is not solidified when heated. Protamine is not a single component, and they are typically a mixture of several components. Different fish species protamine differ greatly in the type and proportion of amino acids, but they also have many similar features, making qualitative and quantitative analysis of protamine sulfate challenging.

The protamine sulfate in the domestic market is mainly extracted from salmonidae and herring species. The quality control of the variety is simpler in the pharmacopoeia of the people's republic of China of 2020 edition, and the peptide map is analyzed by a physical and chemical method which is not carried out effectively. In order to ensure the quality and curative effect of the variety, it is necessary to establish a stable physicochemical analysis method to further perfect the quality control of the variety.

The prior art Hu Chuanmei et al disclose the determination of protamine sulfate content and peptide fragment identification by HPLC combined with LC-MS, the principle being that each peptide in protamine sulfate is separated by liquid phase and then mass spectrometry is performed one by one. In view of the principle of confirming the structure of protamine sulfate by adopting various technical means and the singleness of the prior art means, it is necessary to develop a new detection method to mutually verify the molecular structure of protamine with the technical means disclosed in the prior art.

Disclosure of Invention

The invention aims to provide a novel protamine sulfate detection method, which is used for supplementing structural research of protamine sulfate and increasing similarity research among different samples. The method not only can identify the sequence structure of protamine, but also can compare the similarity between different samples by comparing the proportional relationship between the fragments.

In order to achieve the above purpose, the invention provides a method for detecting a protamine sulfate peptide map, which comprises the following steps:

s1: respectively carrying out enzymolysis on the protamine sulfate bulk drug and a reference preparation and/or a standard substance to obtain a characteristic peptide segment;

s2: and respectively carrying out high performance liquid chromatography tandem mass spectrometry detection on the characteristic peptide fragments from different sources, comparing the retention time, the peak area percentage and the molecular weight of the characteristic peptide Duan Feng, and evaluating the quality of a protamine sulfate sample to be detected.

As an embodiment of the present invention, the protamine sulfate drug substance may be used as a control simultaneously with one or both of the reference formulation or the standard.

As an embodiment of the present invention, the enzyme used in the S1 step may specifically cleave arginine and/or lysine, meaning that cleavage of the enzyme on the carboxy-terminal side of either arginine or lysine has a higher specificity than cleavage on the carboxy-terminal side of any other amino acid. In one aspect, the enzyme specifically cleaves on the carboxy-terminal side of arginine, meaning that the enzyme cleaves on the carboxy-terminal side of arginine with a higher specificity than on the carboxy-terminal side of any other amino acid. In another aspect, the enzyme specifically cleaves on the carboxy-terminal side of lysine, meaning that the enzyme cleaves on the carboxy-terminal side of lysine with a higher specificity than on the carboxy-terminal side of any other amino acid.

The enzyme can be trypsin, endopeptidase Arg-C or carboxypeptidase B. Wherein trypsin is a serine protease and carboxypeptidase B belongs to zinc metalloprotease, and both can specifically cleave carboxyl terminal (or C-terminal) peptide bonds of Lysine (K) and Arginine (R). Endopeptidase Arg-C belongs to serine protease and can specifically cleave the carboxyl terminal peptide bond of arginine. Since protamine has no lysine, trypsin, endopeptidase Arg-C or carboxypeptidase B in the present invention specifically cleaves only the carboxy-terminus of arginine.

As an embodiment of the present invention, the mass ratio of protamine sulfate to enzyme in S1 is 10 to 1000:1, such as further optional 50-200: 1, a step of; the enzymolysis temperature is 35-40 ℃, such as 36 ℃, 37 ℃, 38 ℃ or 39 ℃ further selectable; the enzymolysis time is 10-60 h, such as 16-24 h.

As one embodiment of the present invention, the characteristic peptide consists of 1 to 15 peptide fragments, and the peptide fragments consist of 1 to 4 amino acids, respectively. The peptide amino acid composition and the number of peptide fragments are greatly related to the enzymolysis process, and important parameters in the enzymolysis process include enzymolysis time, the mass ratio of protamine sulfate to enzyme, enzymolysis temperature and the like.

As an embodiment of the present invention, the characteristic peptide fragment may be selected to be PIR, IR, PVR, R, PR, VSR, ASR, GGR, SSR, RPVR, SSSR, RPR, RR, RGGR, RRPR.

As an embodiment of the present invention, the high performance liquid chromatography uses a hydrophilic interaction chromatography column insersil Amide, mobile phase a is an aqueous solution of ammonium acetate or ammonium formate, and mobile phase B is an acetonitrile solution containing an acidic buffer.

The chromatographic column Inertsil Amide uses a chemically stable and tolerant alkylamide (Amide) bonding phase, wherein the matrix is high-purity spherical silica gel.

As an embodiment of the present invention, the mass concentration of ammonium acetate or ammonium formate in the mobile phase A is 1 to 500mM, such as optionally 10 to 100mM.

As one embodiment of the invention, the addition amount of the acid buffer solution in the mobile phase B is 0.01% -1%, such as 0.1% optionally; the acidic buffer is formic acid or acetic acid.

As an embodiment of the present invention, in the high performance liquid chromatography: the flow rate of the mobile phase is 1.0ml/min; column temperature is 40 ℃; the wavelength was UV210nm.

As an embodiment of the present invention, in the high performance liquid chromatography: the elution gradient of mobile phase a and mobile phase B is: as elution proceeds, the addition amount of mobile phase B gradually decreases; in a more specific embodiment, the volume fraction of mobile phase B is reduced from 90% to 55% over 0-35 min; or the volume ratio of the mobile phase B is reduced from 75% to 55% in 0-40 min; or the volume ratio of the mobile phase B is reduced from 75% to 55% in 0-40 min; or the volume ratio of the mobile phase B is reduced from 75% to 65% within 0-40 min.

As an embodiment of the present invention, the mass spectrometry conditions are: ESI ion source, positive ion mode; carrier gas temperature: 350 ℃; drying gas flow rate: 10L/min; capillary voltage: 4000V; mass-to-charge ratio range: 100-1000M/Z.

According to the detection method, the characteristic peptide fragments are obtained by taking the specific enzymolysis protamine as peptide fragments, then the characteristic peptide fragments are subjected to high performance liquid chromatography tandem mass spectrometry HPLC-MS detection, 15 characteristic peptide fragments are separated, excellent retention and separation are realized on a strong polar analyte/mixture which spans a large range in polarity, molecular structure and pKa numerical property, the peak shape of the obtained characteristic peptide fragments is good, and finally the retention time, the peak area percentage and the molecular weight of characteristic peptides Duan Feng from different sources are compared, so that the quality of a protamine sulfate sample to be detected is rapidly estimated, the guidance significance is provided for perfecting the quality control of the variety, and references are provided for deep understanding of the material basis of the variety. The method can be directly converted into high performance liquid chromatography, can also be used for quantitative mass spectrometry for quantitative analysis, and is applied to identification and quality comparison research of protamine sulfate and the like.

Drawings

FIGS. 1 to 3 are chromatograms corresponding to the sample solutions 1 to 3 in example 1, respectively.

FIGS. 4to 6 are chromatograms corresponding to the reference solutions 1 to 3, respectively, in example 1.

FIG. 7 is a chromatogram of the standard solution of example 1.

FIGS. 8 to 22 are mass spectra corresponding to peptide numbers 1 to 15 separated from the sample solution 1 in example 1, respectively.

FIG. 23 is a map corresponding to the peptide PIR, IR, PVR, PR in example 1.

FIG. 24 is a map corresponding to the peptide VSR, ASR, GGR, SSR in example 1.

FIG. 25 is a map corresponding to the peptide RPVR, SSSR, RPR, RR in example 1.

FIG. 26 is a map corresponding to the peptide RGGR and RRPR in example 1.

FIG. 27 is a chromatogram corresponding to the condition of example 2.

FIG. 28 is a chromatogram corresponding to the condition of example 3.

FIG. 29 is a chromatogram corresponding to the condition of example 4.

FIG. 30 is a chromatogram corresponding to the condition of comparative example 1.

FIG. 31 is a chromatogram corresponding to the condition of comparative example 2.

FIG. 32 is a chromatogram corresponding to the condition of comparative example 3.

Detailed Description

In order to make the technical features, objects and advantageous effects of the present invention more clearly understood, the technical solution of the present invention will be described in detail below with reference to the specific embodiments, but the protection content of the present invention is not limited to the following embodiments.

The protamine sulfate bulk drug (extracted from salmon) is derived from pharmaceutical chemicals of beauty star (Nanjing) as a test sample, and a mixture consisting of 4 peptide fragments has the amino acid sequences of:

1PRRRRRSSSRPIRRRRRRRASRRRRRGGRRRR

2PRRRRSSRRPVRRRRRRRVSRRRRRGGRRRR

3PRRRRSSSRPVRRRRRRRVSRRRRRRGGRRRR

4PRRRRASRRIRRRRRRRVSRRRRRGGRRRR。

the protamine sulfate injection adopts a reference preparation (second sixteen batches of reference preparation catalogues) published by a drug review center of the national drug administration, and is derived from Fei Senyou Skabi company (Fresenius Kabi USALLC) as a control.

Trypsin (EC 3.4.21.4) is available from Shanghai Biotechnology Inc.

The protamine sulfate USP standards and other reagents/materials and equipment used in the embodiments are commercially available.

Example 1

Enzyme solution: a trypsin solution was prepared at a concentration of 1mg/ml with 1mM HCl.

Dilution liquid: 63mm NaHCO ₃ Solution, pH 8.5.

Sample stock solution: three batches of protamine sulfate raw material medicines are respectively taken, and a diluent is used for preparing a sample stock solution 1, a sample stock solution 2 and a sample stock solution 3 with the concentration of 5 mg/ml.

Reference stock: three batches of protamine sulfate injection are respectively taken, and after ultrafiltration extraction, a reference stock solution 1, a reference stock solution 2 and a reference stock solution 3 with the concentration of 5mg/ml are prepared by using a diluent.

Standard stock solution: the protamine sulfate standard is taken, and a standard stock solution with the concentration of 5mg/ml is prepared by using a diluent.

Test solution: mixing 500 μl of sample stock solution 1, sample stock solution 2 and sample stock solution 3 with three 50 μl enzyme solutions (the mass ratio of protamine to enzyme is 50:1), and performing enzymolysis at 37deg.C for 16 hr to obtain sample solution 1, sample solution 2 and sample solution 3 containing characteristic peptide fragment.

Reference solution: mixing 500 μl of reference stock solution 1, reference stock solution 2 and reference stock solution 3 with three 50 μl enzyme solutions, and performing enzymolysis at 37deg.C for 16 hr to obtain reference solution 1, reference solution 2 and reference solution 3 containing characteristic peptide fragments.

Standard solution: mixing 500 μl of standard stock solution and 50 μl of enzyme solution, and performing enzymolysis at 37deg.C for 16 hr to obtain standard solution containing characteristic peptide fragment.

High performance liquid chromatography conditions:

chromatographic column: shimadzu Inertsil Amide,3 μm, 4.6X250 mm.

Mobile phase a:100mM ammonium formate; mobile phase B: acetonitrile containing 0.1% formic acid;

the elution gradient is 0-40 min, and the volume ratio of the mobile phase B is reduced from 75% to 65%.

The flow rate of the mobile phase is 1.0ml/min; column temperature is 40 ℃; the wavelength was UV210nm.

Mass spectrometry conditions:

agilent 6224TOF mass spectrometer, ESI ion source, positive ion mode; carrier gas temperature: 350 ℃; drying gas flow rate: 10L/min; capillary voltage: 4000V; mass-to-charge ratio range: 100-1000M/Z.

And respectively carrying out high performance liquid chromatography tandem mass spectrometry detection analysis on 3 batches of sample solutions, 3 batches of reference solutions and 1 batch of standard solution containing the characteristic peptide fragments after trypsin treatment to obtain retention time (specifically shown in the following table 1), peak area percentage (specifically shown in the following table 2) and molecular weight information (specifically shown in the following table 3) of the characteristic peptide Duan Feng. Chromatograms of 3 parts of the sample solution, 3 parts of the reference solution and 1 part of the standard solution are shown in figures 1-7; the mass spectrum of the peptide 1 separated in the sample solution 1 is shown in fig. 8, the mass spectrum of the peptide 2 separated is shown in fig. 9, the mass spectrum of the peptide 3 separated is shown in fig. 10, the mass spectrum of the peptide 4 separated is shown in fig. 11, the mass spectrum of the peptide 5 separated is shown in fig. 12, the mass spectrum of the peptide 6 separated is shown in fig. 13, the mass spectrum of the peptide 7 separated is shown in fig. 14, the mass spectrum of the peptide 8 separated is shown in fig. 15, the mass spectrum of the peptide 9 separated is shown in fig. 16, the mass spectrum of the peptide 10 separated is shown in fig. 17, the mass spectrum of the peptide 11 separated is shown in fig. 18, the mass spectrum of the peptide 12 separated is shown in fig. 19, the mass spectrum of the peptide 13 separated is shown in fig. 20, the peptide 14 separated is shown in fig. 21, and the peptide 15 separated is shown in fig. 22.

And (3) carrying out secondary mass spectrometry (MS 2) analysis by combining a high-resolution mass spectrometer (HRMS), wherein a peptide chain collides with inert gas at high speed in the secondary mass spectrometry to form b/y ions, the amino acid at each position can be confirmed according to the molecular weight difference of the b/y ions by software processing calculation, and the sequence coverage result of a sample is obtained after matching with a theoretical sequence, so that the sequence of a peptide fragment, namely the amino acid composition, is determined, the coverage map of the peptide fragment PIR, IR, PVR, PR is shown in fig. 23, the coverage map of the peptide fragment VSR, ASR, GGR, SSR is shown in fig. 24, the coverage map of the peptide fragment RPVR, SSSR, RPR, RR is shown in fig. 25, and the coverage maps of peptide fragments RGGR and RRPR are shown in fig. 26 (wherein the peptide fragment R of No. 4 is only one amino acid and has no corresponding map).

TABLE 1 Retention time summary table in high Performance liquid chromatography

Evaluation criteria for retention time: the average of retention times for peptide Duan Feng in 3 reference solutions and 1 USP standard was a±0.5min, i.e. lower limit = a-0.5min, upper limit a+0.5min.

TABLE 2 summary of peak area percent in high performance liquid chromatography

Evaluation criteria for percent peak area: peak area percentage extremum (minimum and maximum) R x (1±η) of peptide Duan Feng in 3 reference solutions and 1 USP standard, where η=10%, i.e. lower limit=r _min X (1- η), upper limit=r _max ×(1+η)。

Table 3 mass spectrometry data summary table

According to the analysis of high performance liquid chromatography combined with mass spectrometry, 15 characteristic peptide fragments are obtained after the protamine sulfate is treated by trypsin, and the retention time, the peak area percentage and the molecular weight of each peptide fragment peak in a protamine sulfate sample solution, a reference solution and a USP standard solution are uniform, so that the detection method is suitable for quality identification of the protamine sulfate and has wide application prospect in the field of analysis methods.

Example 2

Controlling the elution gradient of the mobile phase to be 0-35 min in the high performance liquid chromatography condition, wherein the volume ratio of the mobile phase B (acetonitrile containing 0.1% formic acid) is reduced from 90% to 55%; the other conditions are the same as in example 1, and the results are shown in fig. 27, which show that under the gradient condition of the mobile phase, each characteristic peptide segment can be separated, but the baseline is not very stable, and further optimization space exists.

Example 3

Controlling the elution gradient of the mobile phase to be 0-40 min in the high performance liquid chromatography condition, wherein the volume ratio of the mobile phase B (acetonitrile containing 0.1% formic acid) is reduced from 75% to 55%; the other conditions are the same as in example 1, and the results are shown in FIG. 28, which show that under the gradient condition of the mobile phase, each characteristic peptide segment can be separated.

Example 4

The enzymolysis time of the sample solution in the preparation process is 24 hours; controlling the elution gradient of the mobile phase to be 0-40 min in the high performance liquid chromatography condition, wherein the volume ratio of the mobile phase B (acetonitrile containing 0.1% formic acid) is reduced from 75% to 55%; the other conditions were the same as in example 1, and the results showed that 15 characteristic peptide fragments were obtained under the conditions, as shown in FIG. 29. The result is not greatly influenced by enzymolysis for 16 hours and enzymolysis for 24 hours.

According to the research comparison of examples 1-4, it is found that the elution gradient of the mobile phase is controlled in the high performance liquid chromatography conditions, and the separation of protamine can be well realized under the conditions.

Example 5

Mobile phase a in high performance liquid chromatography conditions: 100mM ammonium formate; mobile phase B: acetonitrile containing 0.1% acetic acid; the other conditions are the same as in example 1, and the results show that under the mobile phase condition, after the protamine sulfate is treated by trypsin, the effective separation of 15 characteristic peptide fragments can be realized, so that the method can also be used for directly detecting and comparing the retention time, the peak area percentage and the molecular weight consistency of each peptide fragment peak in a protamine sulfate sample solution, a reference solution and a USP standard solution, and the detection method under the conditions is also suitable for the quality identification of protamine sulfate.

Comparative example 1

Instead of the island body insersil Amide column of example 1, a ACQUITY UPLC BEH HILIC column (using unbound BEH particles, 1.7 μm,3.0 x 150 mm) was used, mobile phase gradient: the volume ratio of mobile phase B was reduced from 100% to 55% for 0-50 min, the other conditions were the same as in example 1, and the results are shown in FIG. 30. The results indicate that under this chromatographic condition, many peptide peaks do not allow for efficient separation. Even if the enzymolysis time is increased from 16h to 22h, detection is carried out again, and the separation result is not improved obviously.

Comparative example 2

Mobile phase a using a Agilent Zorbax Eclipse C18 column (5 μm,4.0×100 mm): 900ml water +100ml methanol +1ml formic acid; mobile phase B:500ml water+500 ml methanol+1 ml formic acid, mobile phase gradient: 0-20 min, the volume ratio of the mobile phase B is increased from 10% to 30%, and the volume ratio of the mobile phase B is increased from 30% to 100% after 20-50 min; the other conditions were the same as in example 1, and the results are shown in FIG. 31. The results indicate that under this chromatographic condition, peptide Duan Feng failed to achieve efficient separation. After the enzymolysis time is increased from 16 hours to 18 hours, detection is carried out again, and no obvious improvement of separation is found.

Through the comparative study of the example 1 and the comparative examples 1-2, chromatographic columns with different action mechanisms, even different types of fillers in the chromatographic columns, are found to have great influence on the separation effect of protamine. The present invention unexpectedly found that the use of a chemically stable, resistant, triple bonded Amide (Amide) bonding phase in a chromatographic column Inertsilamide greatly improved the effective separation of characteristic peptide fragments in protamine relative to a ACQUITY UPLC BEH HILIC chromatographic column. The main reason is that the content of polar amino acids (such as arginine and serine) in protamine is very high, the polarity of peptide fragments after enzymolysis is large, and proper retaining and separating effects cannot be obtained on a C18 column in comparative example 2; the chromatographic column in comparative example 1 cannot obtain good separation effect because of the large number of peptide fragments after enzymolysis and the insignificant polarity difference.

Comparative example 3

The enzymolysis time of the sample solution in the preparation process is 61h; controlling the elution gradient of the mobile phase to be 0-60 min in the high performance liquid chromatography condition, wherein the volume ratio of the mobile phase B (acetonitrile containing 0.1% formic acid) is reduced from 75% to 60%; the other conditions are the same as in example 1, and the result shows that the chromatographic conditions have the advantages of prolonged elution time, reduced gradient and slightly improved separation, but only 14 characteristic peptide fragments are obtained, and the characteristic peptide fragment RGGR (see specifically FIG. 32) is absent, which is presumably because: when the enzymolysis time exceeds 60 hours, RGGR fragments are further subjected to enzymolysis into R and GGR fragments by trypsin, so that the enzymolysis time has a larger influence on result analysis, and when the retention time, the peak area percentage and the molecular weight of each peptide fragment peak in a sample solution of the protamine sulfate, a reference solution and a USP standard solution are compared, the enzymolysis time is strictly controlled and kept consistent, and the accuracy of data is improved.

Claims

1. The protamine sulfate peptide map detection method is characterized by comprising the following steps of:

2. The method of claim 1, wherein: the enzyme used in the step S1 can specifically cleave arginine.

3. The detection method according to claim 2, wherein: the enzyme used in step S1 is selected from trypsin, endopeptidase Arg-C or carboxypeptidase B.

4. A detection method according to any one of claims 1 to 3, wherein: in the step S1, the mass ratio of protamine sulfate to enzyme is 10-1000: 1, the enzymolysis temperature is 35-40 ℃ and the enzymolysis time is 10-60 h.

5. The method of claim 4, wherein: in the step S1, the mass ratio of protamine sulfate to enzyme is 50-200: 1, the enzymolysis temperature is 37 ℃; the enzymolysis time is 16-24 hours.

6. The method of claim 1, wherein: in the step S1, the characteristic peptide segment consists of 1-15 peptide segments, and the peptide segments consist of 1-4 amino acids respectively.

7. The method of detecting according to claim 6, wherein: the characteristic peptide may be PIR, IR, PVR, R, PR, VSR, ASR, GGR, SSR, RPVR, SSSR, RPR, RR, RGGR, RRPR.

8. The method of claim 1, wherein: in the step S2, the high performance liquid chromatography uses a hydrophilic chromatographic column Inertsilamide, the mobile phase A is an aqueous solution of ammonium acetate or ammonium formate, and the mobile phase B is an acetonitrile solution containing an acidic buffer solution.

9. The method of detecting according to claim 8, wherein: in the step S2, the mass concentration of the mobile phase A in the high performance liquid chromatography is 1-500 mM, and the addition amount of the acid buffer solution in the mobile phase B is 0.01% -1%; the acidic buffer is formic acid or acetic acid.

10. The method of claim 1, wherein: in the high performance liquid chromatography: the flow rate of the mobile phase is 1.0ml/min; column temperature is 40 ℃; the wavelength is UV210 nm; the mass spectrum conditions are as follows: ESI ion source, positive ion mode; carrier gas temperature: 350 ℃; drying gas flow rate: 10L/min; capillary voltage: 4000V; mass-to-charge ratio range: 100-1000M/Z.