CN114441658A

CN114441658A - Method for detecting organic acid in bulk drug

Info

Publication number: CN114441658A
Application number: CN202011208621.0A
Authority: CN
Inventors: 张贵民; 刘思光; 李秀艳; 刘忠
Original assignee: Lunan Pharmaceutical Group Corp
Current assignee: Lunan Pharmaceutical Group Corp
Priority date: 2020-11-03
Filing date: 2020-11-03
Publication date: 2022-05-06

Abstract

The invention belongs to the field of drug analysis, and particularly relates to a detection method of organic acid substances in bulk drugs. The invention uses sulfuric acid, phosphoric acid or p-toluenesulfonic acid water solution as mobile phase and uses a high performance liquid chromatograph with an ultraviolet detector or a diode array detector to carry out analysis and detection by connecting a reverse phase chromatographic column and an ion exclusion chromatographic column in series. The method can remove the interference of the matrix on trace organic acid, can quickly and accurately detect the content of the residual organic acid in the raw material medicine, has good linear relation, high precision, high accuracy, good repeatability and durability, high separation degree and strong applicability, is not interfered by the raw material, and fills the blank of the method for measuring the content of the residual organic acid in the raw material medicine.

Description

Method for detecting organic acid in bulk drug

Technical Field

The invention belongs to the field of drug analysis, and particularly relates to a detection method of organic acid substances in bulk drugs.

Background

In the technical field of medicine, most of raw material medicines are obtained by chemical synthesis, in the chemical synthesis, organic acid solvents or by-products of organic acids are mostly used, and in order to ensure the quality of the raw material medicines, the residual quantity of the organic acids in the raw material medicines needs to be detected.

The current methods for detecting organic acids mainly include gas chromatography, liquid chromatography and ion chromatography. In the gas chromatography, since the organic acid substance has a strong activity and is easily adsorbed, the result sensitivity is low and the accuracy is poor. The organic acid is detected by ion chromatography, which is easily interfered by matrix and can not be accurately quantified; and the ion chromatograph has low popularity, thereby further reducing the application of the method. Liquid chromatography is divided into reverse phase chromatography and ion exclusion chromatography, wherein most organic acids are weakly retained, peaks within 5 minutes, and is easily interfered by matrix and mobile phase, and the method has poor durability. Although the traditional ion exclusion chromatography adopts 8% cross-linked resin hydrogen ion chromatographic column for detection, the raw material medicine can also enter the chromatographic column to generate peaks, and the peak shape of the high-content raw material medicine is very wide, so that organic acid with low content can be covered, and the interference of matrix to the organic acid can not be avoided; in addition, the direct entry of the bulk drug into the ion chromatographic column can seriously reduce the service life of the chromatographic column, promote the poor peak shape of the organic acid and reduce the sensitivity. Whether the ion chromatography or the ion exclusion chromatography is used for interfering the matrix, the current common practice is to adopt a solid phase extraction column to pretreat a sample to be tested so that the raw material is adsorbed in the filler, and the interference caused by the subsequent entering of the ion chromatography column or the ion exclusion chromatography column on the detection of the organic acid is avoided. However, the pretreatment process by the solid phase extraction method is complicated, the method needs to be developed and researched, the operation is complicated, the technical requirements are high, and the problems of low recovery rate and poor reproducibility are easily caused.

Therefore, it is necessary to develop a method for detecting an organic acid, which is simple in operation, high in sensitivity, and excellent in accuracy and durability.

Disclosure of Invention

The invention aims to provide a high performance liquid chromatography for detecting organic acid residues in bulk drugs, which effectively separates the bulk drugs from organic acids by combining a reversed phase chromatographic column and an ion exclusion chromatographic column so as to quickly detect the organic acid residues in the bulk drugs and ensure stable and controllable quality of the bulk drugs.

The technical scheme of the invention is as follows:

a method for detecting organic acid in bulk drug adopts a reversed-phase chromatographic column connected in series with an ion exclusion chromatographic column, takes sulfuric acid, phosphoric acid or p-toluenesulfonic acid aqueous solution as a mobile phase, and carries out analysis and detection by a high performance liquid chromatograph with an ultraviolet detector or a diode array detector.

Preferably, the reverse phase chromatographic column is a C8 column, a C18 column, a phenyl column, an amino column, a pentafluorophenyl column, a cyano column or a bare silica gel column, and more preferably a C18 column. The present invention is not particularly limited as to the type of reverse phase chromatography column, for example in some embodiments the reverse phase chromatography column is phenomenex onyx monolithinic HD-C18, in other embodiments the reverse phase chromatography column is YMC Triart C18, and in other embodiments the reverse phase chromatography column is Welch XB-C18.

Preferably, the ion exclusion chromatographic column is a cross-linked resin hydrogen ion chromatographic column, more preferably a cross-linked sulfonated styrene-divinylbenzene hydrogen ion chromatographic column, and further more preferably, the ion exclusion chromatographic column is a cross-linked sulfonated styrene-divinylbenzene hydrogen ion chromatographic column with cross-linking degrees of 5%, 8% and 10%. The invention is not particularly limited as to the type of ion exclusion chromatography column, for example in some embodiments the ion exclusion chromatography column is Rezex ROA-Organic Acid H +, in other embodiments the ion exclusion chromatography column is Aminex HPX-87H, and in other embodiments the ion exclusion chromatography column is Shim-pack SCR-102H.

Preferably, the mobile phase is 0.1-10 mmol/L sulfuric acid aqueous solution, phosphoric acid aqueous solution and p-toluenesulfonic acid aqueous solution; preferably 5-10 mmol/L sulfuric acid water solution; further, the mobile phase is 8-10 mmol/L sulfuric acid aqueous solution. In some embodiments, the mobile phase is an 8mmol/L aqueous sulfuric acid solution; in other embodiments, the mobile phase is a 9mmol/L aqueous sulfuric acid solution; in other embodiments, the mobile phase is a 10mmol/L aqueous sulfuric acid solution.

Preferably, the flow rate of the mobile phase is 0.4 to 0.8ml/min, and more preferably 0.5 to 0.6 ml/min.

Preferably, the temperature of the chromatographic column is 30-60 ℃, and preferably 40-45 ℃.

Preferably, the detection wavelength is 200-210 nm.

Preferably, the sample injection amount is 10-30 mu l.

Preferably, the organic acid includes, but is not limited to, formic acid, acetic acid, butyric acid, malonic acid, citric acid, succinic acid, tartaric acid, malic acid, fumaric acid, succinic acid, ascorbic acid, oxalic acid, lactic acid, benzoic acid, and the like.

The invention does not limit the category of the raw material medicine, and the detection can be carried out by the technical scheme of the invention as long as the synthesis process of the raw material medicine uses organic acid and the raw material medicine possibly has organic acid.

The following contents take formic acid, acetic acid, malonic acid, citric acid, tartaric acid and fumaric acid as examples to illustrate the detection method, and the method for measuring the organic acid in the bulk drugs can be realized by the following steps:

(1) preparation of the solution

Comparison product stock solution: taking proper amount of formic acid, acetic acid, malonic acid, citric acid and tartaric acid, respectively and precisely weighing, and respectively diluting with diluent to about 500 mu g/ml, taking proper amount of fumaric acid, precisely weighing, and diluting with diluent to about 100 mu g/ml.

Sample solution: taking a proper amount of the raw material medicines, precisely weighing, dissolving by using a diluent, and diluting to prepare a solution containing 10mg/ml of the raw material medicines.

Mixing the reference solution: taking a proper amount of formic acid, acetic acid, malonic acid, citric acid and tartaric acid, precisely weighing, and diluting with a diluent to prepare a solution of about 50 mu g/ml; taking a proper amount of fumaric acid, precisely weighing, and diluting with a diluent to prepare a solution with the concentration of about 10 mu g/ml;

adding a standard solution into a sample: taking a test sample and a reference sample, precisely weighing, dissolving with a reference solution, and diluting.

(2) Chromatography system detection

And respectively injecting the reference substance solution, the test substance solution, the mixed reference substance solution and the test substance added standard solution into a chromatograph for detection, and calculating the acid residue in the test substance by an external standard method according to the peak area. The detection conditions were as follows:

a chromatographic column: the reverse phase chromatographic column is connected with the ion exclusion chromatographic column in series;

temperature of the column: 40-45 ℃;

mobile phase: 0.1-10 mmol/L sulfuric acid water solution;

flow rate of mobile phase: 0.4 to 0.8ml/min

Detection wavelength: 210 nm;

sample introduction amount: 10 to 20 μ l.

Preferably, the diluent in the solution preparation is a mobile phase or purified water.

Preferably, the mobile phase is a 5-10 mmol/L sulfuric acid aqueous solution.

Preferably, the flow rate of the mobile phase is 0.5-0.6 ml/min.

Preferably, the sample size is 20. mu.l.

The detection method is suitable for detecting organic acids which are used in a raw material medicine synthesis or preparation method and can cause the organic acids to be remained in the raw material medicines, such as zolpidem tartrate, amlodipine besylate, afatinib maleate, isosorbide mononitrate, ketoprofen tromethamine, riluzole, gimeracil and the like.

According to the method, the reverse phase chromatographic column and the ion exclusion chromatographic column are connected in series, so that the interference of the matrix on trace organic acid can be removed, the content of the residual organic acid in the raw material medicine can be rapidly and accurately detected, the linear relation is good, the precision is high, the accuracy is high, the repeatability and the durability are good, the separation degree is high, the applicability is strong, the interference of the raw material is avoided, and the blank of the method for determining the content of the residual organic acid in the raw material medicine is filled.

Drawings

FIG. 1: example 1 tartaric acid control profile;

FIG. 2: example 1 citric acid control profile;

FIG. 3: example 1 malonic acid control profile;

FIG. 4: example 1 formic acid control profile;

FIG. 5: example 1 acetic acid control profile;

FIG. 6: example 1 fumaric acid control profile;

FIG. 7: example 1 mixed control profile;

FIG. 8: example 1 test article solution profile;

FIG. 9: example 1 sample spiking solution profile;

FIG. 10: example 2 mixing control localization profiles;

FIG. 11: example 2 test article solution profile;

FIG. 12: example 3 test sample spiking solution atlas;

FIG. 13: example 4 test sample spiking solution profile;

FIG. 14: example 5 mixed control localization profiles;

FIG. 15: example 5 test article solution profile;

FIG. 16: example 6 mixed control localization profiles;

FIG. 17: example 6 test article solution profile;

FIG. 18: example 7 mixed control localization profiles;

FIG. 19: example 7 test article solution profile;

FIG. 20: sensitivity solution profile.

Detailed Description

The present invention is further explained by reference to the following specific examples, which should be understood that the following examples are only for illustrating the technical solutions of the present invention and are not intended to limit the scope of the present invention. The raw material medicaments can be prepared by self synthesis according to the method of the prior art and can also be obtained by commercial means.

Example 1: determination of formic acid residue in zolpidem tartrate bulk drug

(1) Solution preparation

Preparing a reference substance stock solution: 13.05mg of formic acid, 14.61mg of acetic acid, 12.71mg of malonic acid, 13.08mg of tartaric acid and 12.44mg of citric acid are accurately weighed in a 25ml volumetric flask, 5.033mg of fumaric acid is accurately weighed in a 50ml volumetric flask, and the mobile phase is dissolved as a diluent and the volume is determined.

Mix control stock solution: accurately weighing 13.01mg of formic acid, 14.66mg of acetic acid, 12.73mg of malonic acid, 13.05mg of tartaric acid and 12.47mg of citric acid into a 25ml volumetric flask, dissolving the fumaric acid reference solution and fixing the volume to obtain a 10-time reference stock solution.

Control localization solution: taking 1ml of the stock solution of each reference substance in a 10ml volumetric flask, diluting with a mobile phase and fixing the volume.

Mixing the reference solution: taking 5ml of the mixed reference stock solution, diluting with a diluent and fixing the volume to 50 ml.

Test solution: 100.70mg of zolpidem tartrate is precisely weighed into a 10ml volumetric flask, diluted with a mobile phase and fixed to the volume.

Adding a standard solution into a test sample: 100.40mg of zolpidem tartrate is precisely weighed into a 10ml volumetric flask, diluted by a reference substance stock solution and subjected to constant volume.

(2) Chromatography system detection

And respectively injecting the reference substance positioning solution, the mixed reference substance solution, the test sample solution and the test sample added standard solution into a chromatographic system, recording a chromatogram, determining the nature of the retention time of organic acid in the reference substance positioning solution, and calculating the formic acid residue content in the zolpidem tartrate test sample by peak area according to an external standard method.

The instrument comprises the following steps: waters e2695 liquid chromatography system;

a chromatographic column: phenomenex onyx monolithinic HD-C18, 100X 4.6mm tandem Rezex ROA-Organic Acid H + (8%), 300X 7.8 mm;

mobile phase: 9mmol/L sulfuric acid water solution;

flow rate: 0.6 ml/min;

detection wavelength: 210 nm;

column temperature: 40 ℃;

sample introduction amount: 20 μ l

The reference substance location map is shown in figures 1-6, the mixed reference substance map is shown in figure 7, the test substance map is shown in figure 8, and the test substance labeling map is shown in figure 9. As can be seen from fig. 1 to 6, the retention time of tartaric acid was 12.130min, the retention time of citric acid was 13.079min, the retention time of malonic acid was 14.251min, the retention time of formic acid was 17.385min, the retention time of acetic acid was 19.289min, and the retention time of fumaric acid was 20.975 min; as can be seen from fig. 7, tartaric acid, citric acid, malonic acid, formic acid, acetic acid and fumaric acid show peaks according to the above, and the chromatographic peaks of the organic acids can be effectively separated, so that the separation degree is good; from FIG. 8, it can be seen that zolpidem tartrate contains no residual formic acid in the bulk drug.

Example 2 determination of formic acid residue in zolpidem tartrate drug substance

The mixed reference solution and the sample solution prepared in example 1 were injected into the following chromatographic system, chromatograms were recorded, retention time of organic acid in the reference localization solution was used for qualitative determination, and the formic acid residue content in the zolpidem tartrate sample was calculated by peak area according to the external standard method.

Detection of a chromatographic system:

a chromatographic column: phenomenex onyx monolithic HD-C18, 100X 4.6mm tandem Rezex ROA-Organic Acid H + (8%), 300X 7.8 mm;

mobile phase: 10mmol/L sulfuric acid water solution;

flow rate: 0.5 ml/min;

detection wavelength: 210 nm;

column temperature: 50 ℃;

sample introduction amount: 20 μ l

The reference substance positioning spectrum is shown in figure 10, the sample spectrum is shown in figure 11, and as can be seen from figure 10, the chromatographic peaks of the organic acids can be effectively separated, and the separation degree is good; as can be seen from FIG. 11, the zolpidem tartrate bulk drug does not contain any residual formic acid.

EXAMPLE 3 determination of formic acid residue in zolpidem tartrate drug substance

Preparing a sample adding solution according to the method of the embodiment 1, injecting the sample adding solution into a chromatographic system respectively, and recording the chromatogram.

A chromatographic system:

the instrument comprises the following steps: an Ultimate 3000 liquid chromatography system;

and (3) chromatographic column: phenomenex onyx monolithinic HD-C18, 100X 4.6 mm;

mobile phase: 9mmol/L sulfuric acid water solution;

flow rate: 0.6 ml/min;

detection wavelength: 210 nm;

column temperature: 40 ℃;

sample introduction amount: 20 μ l

The chromatogram of the sample solution is shown in FIG. 12. As can be seen from FIG. 12, the retention of organic acid was weak and the peak time was early, which caused matrix interference and failed to achieve effective separation.

Example 4 determination of formic acid residue in zolpidem tartrate bulk drug

The sample solutions prepared by the method of example 1 are injected into the chromatographic system respectively, and the chromatograms are recorded.

A chromatographic system:

a chromatographic column: rezex ROA-Organic Acid H + (8%), 300X 7.8 mm;

mobile phase: 9mmol/L sulfuric acid water solution;

flow rate: 0.6 ml/min;

detection wavelength: 210 nm;

column temperature: 40 ℃;

sample introduction amount: 20 μ l

The chromatogram of the sample solution is shown in FIG. 13. As can be seen from fig. 13, the concentration of the sample was high, the response was high, and the detection of organic acid was easily interfered.

Example 5 detection of acetic acid residue in isosorbide mononitrate drug substance

The organic acid control mixed solution was prepared in the same manner as in example 1.

Test solution: isosorbide mononitrate 100.27mg was weighed out accurately and diluted to 10ml with mobile phase.

And respectively injecting the reference substance mixed solution and the test sample solution into a chromatographic system, recording a chromatogram, and calculating the acetic acid residue content in the isosorbide mononitrate test sample by using a peak area according to an external standard method.

a chromatographic column: YMC Triart C18,4.6X150mm,5 μm tandem Rezex ROA-Organic Acid H + (8%), 300X 7.8 mm;

mobile phase: 1mmol/L sulfuric acid water solution;

flow rate: 0.4 ml/min;

detection wavelength: 210 nm;

column temperature: 40 ℃;

sample introduction amount: 20 μ L

The mixed reference substance spectrum is shown as 14, the sample spectrum is shown as 15, and as can be seen from fig. 14, the chromatographic peaks of the organic acids can be effectively separated, and the separation degree is good; from fig. 15, it can be seen that isosorbide mononitrate drug substance contains no acetic acid residue.

Example 6 detection of acetic acid residue in gimeracil drug substance

The control mixed solution was prepared in the same manner as in example 1.

Test solution: precisely weighing 100.13mg of gimeracil in a 10ml volumetric flask, diluting with a mobile phase and fixing the volume.

And respectively injecting the reference substance positioning solution, the test sample solution and the test sample added standard solution into a chromatographic system, recording a chromatogram, determining the retention time of the organic acid in the reference substance mixed solution, and calculating the acetic acid residue content in the gimeracil test sample by peak area according to an external standard method.

The instrument comprises: an Ultimate 3000 liquid chromatography system;

a chromatographic column: welch XB-C18, 150X4.6mm, 5 μm in tandem Aminex HPX-87H (8%), 300X 7.8 mm;

mobile phase: 5mmol/L phosphoric acid water solution;

flow rate: 0.8 ml/min;

detection wavelength: 210 nm;

column temperature: 40 ℃;

sample introduction amount: 20 μ l

The mixed reference substance spectrum is shown as 16, the sample spectrum is shown as 17, and as can be seen from fig. 16, the chromatographic peaks of the organic acids can be effectively separated, and the separation degree is good; as can be seen from fig. 17, gemfibrozil drug substance contained no acetic acid residue.

Example 7 determination of acetic acid residue in riluzole drug substance

A control mixed solution was prepared in accordance with the method of example 1.

Test solution: riluzole 100.13mg was weighed precisely into a 10ml volumetric flask, diluted with mobile phase and made to volume.

And respectively injecting the reference substance positioning solution, the test sample solution and the test sample labeling solution into a chromatographic system, recording a chromatogram, determining the retention time of the organic acid in the reference substance mixed solution, and calculating the acetic acid residue content in the riluzole test sample by peak area according to an external standard method.

mobile phase: 8mmol/L sulfuric acid water solution;

flow rate: 0.5 ml/min;

detection wavelength: 210 nm;

column temperature: 40 ℃;

sample introduction amount: 20 μ l

The mixed reference substance spectrum is shown as 18, the sample spectrum is shown as 19, and as can be seen from the graph 18, the chromatographic peaks of the organic acids can be effectively separated, and the separation degree is good; it can be seen from figure 19 that riluzole drug substance contains no acetic acid residue.

Example 8 verification of examples

(1) System precision detection

The preparation method of the zolpidem tartrate sample and standard solution is the same as that in example 1, the sample and standard solution is used as a system applicability solution, 20 mu l of the system applicability solution is precisely measured and injected into a liquid chromatograph, 6 needles are continuously injected, the measurement is carried out according to the chromatographic conditions in example 1, chromatograms are recorded, and the RSD of the organic acid peak area of each chromatogram, the formic acid retention time and the peak area result are respectively calculated and shown in Table 1.

TABLE 1

As can be seen from Table 1, the peak area RSD of each organic acid is less than 1%, and it can be seen that the system precision is good

(2) Linear detection of organic acids

The preparation method of the mixed reference stock solution is the same as that in example 1, the mixed reference stock solutions are respectively placed in 10ml measuring bottles in precise amount, 500 mu L, 800 mu L, 1ml, 1.2ml, 1.5ml and 2ml, and the mobile phase is added to dilute the mixed reference stock solutions to the scale to prepare fumaric acid with the concentration of 5 mu g/L, 8 mu g/L, 10 mu g/L, 12 mu g/L, 15 mu g/L and 20 mu g/L; formic acid, acetic acid, malonic acid, tartaric acid and citric acid, wherein the five organic acids are 25 mu g/L, 40 mu g/L, 50 mu g/L, 60 mu g/L, 75 mu g/L and 100 mu g/L of the linear solution. Precisely measuring 20 μ l of each linear solution, injecting into a liquid chromatograph, detecting according to the chromatographic conditions of example 1, recording the chromatogram, and performing linear regression with the concentration mg/ml as abscissa and the peak area (mAu × min) as ordinate to obtain the linear regression equation of each organic acid as shown in table 2.

TABLE 2 Linear test results of organic acids

As can be seen from the results in Table 2, the linear relationship between the peak area and the concentration of each component is good in the concentration range of 0.005 mg/ml-0.02 mg/ml fumaric acid and 0.025 mg/ml-0.1 mg/ml organic acid.

(3) Sensitive detection of organic acids

The preparation method of the organic acid reference stock solution is the same as that in example 1, 500. mu.l of the fumaric acid stock solution is precisely measured and placed in a 10ml volumetric flask, and the solution is diluted by a mobile phase and subjected to volume fixing to be used as a fumaric acid mother solution. 100 mul of tartaric acid stock solution, 200 mul of citric acid stock solution and malonic acid stock solution, 250 mul of formic acid stock solution, 500 mul of acetic acid stock solution and 200 mul of fumaric acid mother solution are respectively taken in a 10ml volumetric flask, diluted by a mobile phase and subjected to constant volume to be used as a sensitivity solution. 20. mu.l of the sensitive solution was measured precisely, and the solution was injected into a liquid chromatograph, detected under the chromatographic conditions of example 1, and the spectrum was recorded. As shown in FIG. 20, the S/N ratio of each organic acid was about 15, indicating that the detection method of the present invention has high sensitivity.

(4) Accurate detection of organic acid substance content

The preparation methods of the reference stock solution, the sample solution and the mixed reference solution are the same as those in example 1.

50% accuracy test solution: accurately weighing 100.0mg zolpidem tartrate in a 10ml volumetric flask, adding 0.5ml of reference substance stock solution, diluting to scale with mobile phase, and shaking up to obtain the final product; 3 parts of the raw materials are prepared by the same method.

100% accuracy test solution: accurately weighing 100.1mg zolpidem tartrate in a 10ml volumetric flask, adding 1ml of reference stock solution, diluting to scale with mobile phase, and shaking up to obtain 3 parts by the same method.

150% accuracy test solution: accurately weighing 100.0mg of zolpidem tartrate in a 10ml volumetric flask, adding 1.5ml of reference stock solution, diluting to scale with a mobile phase, and shaking up to obtain 3 parts by the same method.

The sample solution, the mixed control solution, the 50% accuracy test solution, the 100% accuracy test solution, and the 150% accuracy test solution were each measured precisely by 20 μ l, and each solution was injected into a liquid chromatograph, measured under the chromatographic conditions of example 1, and a chromatogram was recorded, and the recovery rate, the average recovery rate (n ═ 9), and the RSD of each organic acid in the three concentration test solutions were calculated by peak area according to an external standard method. The results are shown in Table 3.

TABLE 3 organic acid content detection accuracy test results

As can be seen from Table 3, the recovery rates of the organic acids are all more than 99%, and the RSD is less than 3%, indicating that the detection method of the invention has good accuracy.

Claims

1. A method for detecting organic acid in bulk drugs is characterized in that a reversed-phase chromatographic column is adopted and connected with an ion exclusion chromatographic column in series, sulfuric acid, phosphoric acid or p-toluenesulfonic acid aqueous solution is used as a mobile phase, and a high performance liquid chromatograph with an ultraviolet detector or a diode array detector is used for analysis and detection.

2. The method of claim 1, wherein the reverse phase chromatography column is a C18 column, a C8 column, a phenyl column, an amino column, a pentafluorophenyl column, a cyano column, or a bare silica gel column.

3. The method of claim 1, wherein the ion exclusion chromatography column is a cross-linked resin hydrogen ion chromatography column, preferably a cross-linked sulfonated styrene-divinylbenzene hydrogen ion chromatography column.

4. The method of claim 3, wherein the ion exclusion chromatography column has a degree of cross-linking of 5%, 8%, or 10%.

5. The method according to claim 1, wherein the concentration of the mobile phase is 0.1 to 10 mmol/L.

6. The method according to claim 1, wherein the mobile phase is 5-10 mmol/L sulfuric acid aqueous solution.

7. The method according to claim 1, wherein the flow rate of the mobile phase is 0.4 to 0.8 ml/min.

8. The method according to claim 1, wherein the column temperature is 30 to 60 ℃.

9. The method of claim 1, wherein the detection wavelength is 200-210 nm.

10. The method according to claim 1, wherein the amount of the sample is 10 to 30. mu.l.