CN114441658A - Method for detecting organic acid in bulk drug - Google Patents

Abstract

The invention belongs to the field of drug analysis, and particularly relates to a method for detecting organic acids in raw materials. In the present invention, a reversed-phase chromatographic column and an ion-exclusion chromatographic column are connected in series, and the aqueous solution of sulfuric acid, phosphoric acid or p-toluenesulfonic acid is used as the mobile phase, and the analysis and detection are carried out by a high-performance liquid chromatograph with an ultraviolet detector or a diode array detector. The method of the invention can remove the interference of the matrix to the trace organic acid, can quickly and accurately detect the content of the residual organic acid in the raw material medicine, has good linear relationship, high precision, high accuracy, good repeatability and durability, and high separation degree , strong applicability, free from the interference of raw materials, filling the blank of the method for the determination of residual organic acid content in raw materials.

Description

A kind of detection method of organic acid in raw material medicine

technical field

The invention belongs to the field of drug analysis, and particularly relates to a method for detecting organic acids in raw materials.

Background technique

In the field of pharmaceutical technology, most APIs are obtained by chemical synthesis. In chemical synthesis, most organic acid solvents or by-products that produce organic acids are used. In order to ensure the quality of APIs, it is necessary to detect APIs. residues of these organic acids.

At present, the methods for detecting organic acids mainly include gas chromatography, liquid chromatography, and ion chromatography. In gas chromatography, due to the strong activity of organic acid substances, adsorption is easy to occur, resulting in low sensitivity and poor precision of results. The detection of organic acids by ion chromatography is easy to be interfered by the matrix and cannot be accurately quantified; and the low penetration rate of ion chromatography further reduces the application of such methods. Liquid chromatography is further divided into reversed-phase chromatography and ion exclusion chromatography. In reversed-phase chromatography, most organic acids have weak retention, and peaks appear within 5 minutes, which are easily interfered by the matrix and mobile phase. The method is less durable. Although the traditional ion exclusion chromatography uses an 8% cross-linked resin hydrogen ion chromatographic column for detection, the API will also enter the chromatographic column and produce peaks. In addition, the direct entry of the API into the ion chromatographic column will seriously reduce the life of the chromatographic column, cause the peak shape of the organic acid to deteriorate, and reduce the sensitivity. Regardless of ion chromatography or ion exclusion chromatography, for the interference of the matrix, the current common practice is to use a solid-phase extraction column to pre-treat the test sample, so that the raw material is adsorbed in the filler and avoids subsequent entry into the ion chromatography column or ions. The size exclusion column in turn interferes with the detection of organic acids. However, the pretreatment process of the solid-phase extraction method is relatively complicated, the method exploration requires further development and exploration, the operation is relatively complicated, the technical requirements are high, and the problems of low recovery rate and poor reproducibility are prone to occur.

Therefore, it is necessary to develop an organic acid detection method with simple operation, high sensitivity, high accuracy and good durability.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a high-performance liquid chromatography method for the detection of organic acid residues in raw materials. Through the combination of reversed-phase chromatography column and ion exclusion chromatography column, the raw materials and organic acids can be effectively separated to rapidly detect raw materials. The residual amount of organic acids in the medicine makes the quality of the API stable and controllable.

The technical scheme of the present invention is as follows:

A method for detecting organic acids in raw materials, which adopts a reversed-phase chromatographic column in series with an ion-exclusion chromatographic column, uses sulfuric acid, phosphoric acid or an aqueous solution of p-toluenesulfonic acid as a mobile phase, and uses a high-efficiency detector with an ultraviolet detector or a diode array detector. Liquid chromatograph for analysis and detection.

Preferably, the reversed-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, more preferably a C18 column. The present invention does not specifically limit the type of reversed-phase chromatography column, for example, in some embodiments, the reversed-phase chromatography column is phenomenex onyx monolitnic HD-C18, in other embodiments, the reversed-phase chromatography column is YMCTriart C18, in other embodiments The reversed-phase column was 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, the ion exclusion chromatographic column is a cross-linking degree 5%, 8%, 10% cross-linked sulfonated styrene-divinylbenzene hydrogen ion chromatography column. The present invention does not specifically limit 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, in other embodiments In some 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, p-toluenesulfonic acid aqueous solution; preferably 5-10 mmol/L sulfuric acid aqueous solution; further mobile phase is 8-10 mmol/L sulfuric acid aqueous solution . In some embodiments, the mobile phase is an aqueous sulfuric acid solution of 8 mmol/L; in other embodiments, the mobile phase is an aqueous sulfuric acid solution of 9 mmol/L; in other embodiments, the mobile phase is an aqueous sulfuric acid solution of 10 mmol/L.

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

Preferably, the temperature of the chromatographic column is 30-60°C, preferably 40-45°C.

Preferably, the detection wavelength is 200-210 nm.

Preferably, the injection volume is 10-30 μl.

Preferably, the organic acids include but are 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 other organic acids.

The present invention does not limit the category of the bulk drug, as long as the organic acid is used in the synthesis process of the bulk drug, there may be organic acids in the bulk drug, which can be detected by the technical solution of the present invention.

The following content takes formic acid, acetic acid, malonic acid, citric acid, tartaric acid, and fumaric acid as examples to illustrate the detection method of the present invention, and a method for the determination of organic acids in a crude drug can be achieved through the following steps:

(1) Preparation of solution

①Reference stock solution: Take appropriate amounts of formic acid, acetic acid, malonic acid, citric acid, and tartaric acid, accurately weigh them, and dilute them with diluents to about 500 μg/ml. Take an appropriate amount of fumaric acid, accurately weigh, and dilute with The solution was diluted to a solution of about 100 μg/ml.

②Test solution: Take an appropriate amount of the API, accurately weigh it, dissolve and dilute it with a diluent to make a solution containing 10mg/ml of the API.

③ Mixed reference solution: Take appropriate amount of formic acid, acetic acid, malonic acid, citric acid, and tartaric acid, accurately weigh, and dilute with diluent to make a solution of about 50 μg/ml; take an appropriate amount of fumaric acid, accurately weigh, and dilute with The liquid is diluted to make a solution of about 10 μg/ml;

④ Standard solution for the test substance: Take the test substance and reference substance, accurately weigh them, dissolve and dilute them with the reference substance solution.

(2) Chromatographic system detection

The above-mentioned reference solution, test solution, mixed reference solution, and test solution were respectively injected into the chromatograph for detection, and the acid residue in the test was calculated by the peak area of the external standard method. The detection conditions are as follows:

Chromatographic column: reversed-phase chromatographic column in series with ion exclusion chromatographic column;

Column temperature: 40～45℃;

Mobile phase: 0.1～10mmol/L sulfuric acid aqueous solution;

Mobile phase flow rate: 0.4～0.8ml/min

Detection wavelength: 210nm;

Injection volume: 10～20μl.

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

Preferably, the mobile phase is 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 injection volume is 20 μl.

The detection method of the present invention is suitable for the detection of organic acids used in the synthesis or preparation method of raw materials, which may cause organic acids to remain in the raw materials, such as zolpidem tartrate, amlodipine besylate, afatinib maleate Detection of organic acids in raw materials such as nidrolone, isosorbide mononitrate, tromethamine, riluzole, gimeracil, etc.

By connecting a reversed-phase chromatographic column and an ion-exclusion chromatographic column in series, the invention can remove the interference of the matrix on the trace organic acid, can quickly and accurately detect the content of the residual organic acid in the crude drug, has good linear relationship, high precision and high accuracy , Good repeatability and durability, high degree of separation, strong applicability, free from the interference of raw materials, filling the blank of the method for the determination of residual organic acid content in raw materials.

Description of drawings

Fig. 1: embodiment 1 tartaric acid reference substance spectrum;

Fig. 2: embodiment 1 citric acid reference substance spectrum;

Fig. 3: embodiment 1 malonic acid reference substance spectrum;

Fig. 4: embodiment 1 formic acid reference substance spectrum;

Fig. 5: embodiment 1 acetic acid reference substance spectrum;

Fig. 6: embodiment 1 fumaric acid reference substance spectrum;

Fig. 7: embodiment 1 mixed reference substance spectrum;

Fig. 8: embodiment 1 need testing solution spectrum;

Fig. 9: embodiment 1 test sample spiked solution spectrum;

Figure 10: The location map of the mixed reference substance in Example 2;

Fig. 11: embodiment 2 need testing solution spectrum;

Figure 12: embodiment 3 test sample spiked solution spectrum;

Figure 13: embodiment 4 test sample spiked solution spectrum;

Figure 14: The location map of the mixed reference substance in Example 5;

Figure 15: embodiment 5 need testing solution spectrum;

Figure 16: The location map of the mixed reference substance in Example 6;

Figure 17: embodiment 6 need testing solution spectrum;

Fig. 18: The location map of embodiment 7 mixed reference substance;

Figure 19: embodiment 7 need testing solution spectrum;

Figure 20: Sensitivity solution profile.

Detailed ways

The present invention will be further explained below in conjunction with specific embodiments, it should be understood that the following embodiments are only used to illustrate the technical solutions of the present invention, and are not used to limit the protection scope of the present invention. The API can be synthesized and prepared according to the methods of the prior art, and can also be obtained through commercial channels.

Embodiment 1: Determination of formic acid residues in Zolpidem tartrate crude drug

(1) Solution preparation

Preparation of reference stock solution: Accurately weigh 13.05 mg of formic acid, 14.61 mg of acetic acid, 12.71 mg of malonic acid, 13.08 mg of tartaric acid, and 12.44 mg of citric acid into a 25ml volumetric flask, and accurately weigh 5.033mg of fumaric acid into a 50ml volumetric flask , the mobile phase was dissolved and made up to volume as a diluent.

Mixed reference substance stock solution: Accurately weigh 13.01 mg of formic acid, 14.66 mg of acetic acid, 12.73 mg of malonic acid, 13.05 mg of tartaric acid, and 12.47 mg of citric acid into a 25 ml volumetric flask, dissolve and dilute with the above-mentioned fumaric acid reference solution, That is, a 10-fold reference stock solution is obtained.

Reference substance positioning solution: Take 1ml of the above reference substance stock solutions in a 10ml volumetric flask, dilute with mobile phase and make up to volume.

Mixed reference substance solution: Take 5ml of the above mixed reference substance stock solution, dilute with diluent and dilute to 50ml.

Test solution: Accurately weigh 100.70 mg of Zolpidem tartrate in a 10 ml volumetric flask, dilute with mobile phase and make up to volume.

Standard solution for the test substance: Accurately weigh 100.40 mg of Zolpidem tartrate in a 10 ml volumetric flask, dilute it with the reference substance stock solution and make up to volume.

(2) Chromatographic system detection

Inject the above-mentioned reference substance locating solution, mixed reference substance solution, test solution, and test sample addition solution into the chromatographic system respectively, record the chromatogram, and qualitatively determine the retention time of the organic acid in the reference substance locating solution according to the external standard method. The residual formic acid content in the test sample of Zolpidem tartrate was calculated by the peak area.

Instrument: Waters e2695 liquid chromatography system;

Chromatographic column: phenomenex onyx monolitnic HD-C18, 100×4.6mm series Rezex ROA-Organic Acid H+ (8%), 300×7.8mm;

Mobile phase: 9mmol/L sulfuric acid aqueous solution;

Flow rate: 0.6ml/min;

Detection wavelength: 210nm;

Column temperature: 40℃;

Injection volume: 20μl

The map of the reference substance is shown in Figure 1-6, the map of the mixed reference substance is shown in Figure 7, the map of the test product is shown in Figure 8, and the map of the test product is shown in Figure 9. As can be seen from Figure 1-6, the retention time of tartaric acid is 12.130min, the retention time of citric acid is 13.079min, the retention time of malonic acid is 14.251min, the retention time of formic acid is 17.385min, the retention time of acetic acid is 19.289 The retention time of min and fumaric acid is 20.975min; as can be seen from Figure 7, tartaric acid, citric acid, malonic acid, formic acid, acetic acid, and fumaric acid appear peaks accordingly, and the chromatographic peaks of each organic acid can be effectively separated and separated. It can be seen from Figure 8 that there is no formic acid residue in the zolpidem tartrate API.

Embodiment 2 Determination of formic acid residues in Zolpidem tartrate crude drug

The mixed reference substance solution and the need testing solution prepared in Example 1 were injected into the following chromatographic system, the chromatogram was recorded, and the retention time of the organic acid in the reference substance was determined by the retention time of the organic acid in the solution, and Zolpidem tartrate was calculated by the external standard method with the peak area. Formic acid residual content in the test sample.

Chromatographic system detection:

Instrument: Waters e2695 liquid chromatography system;

Chromatographic column: Phenomenex onyx monolitnic HD-C18, 100×4.6mm series Rezex ROA-Organic Acid H+ (8%), 300×7.8mm;

Mobile phase: 10mmol/L sulfuric acid aqueous solution;

Flow rate: 0.5ml/min;

Detection wavelength: 210nm;

Column temperature: 50℃;

Injection volume: 20μl

The reference substance location map is shown in Figure 10, and the test sample map is shown in Figure 11. It can be seen from Figure 10 that the chromatographic peaks of each organic acid can be effectively separated and the separation is good; it can be seen from Figure 11 that there is no difference in the zolpidem tartrate API. Contains formic acid residues.

Embodiment 3 Determination of formic acid residues in Zolpidem tartrate crude drug

According to the method of Example 1, the standard addition solution of the test sample was prepared, and the standard addition solution of the test sample was injected into the chromatographic system respectively, and the chromatogram was recorded.

Chromatography system:

Instrument: Ultimate 3000 liquid chromatography system;

Chromatographic column: phenomenex onyx monolitnic HD-C18, 100×4.6mm;

Mobile phase: 9mmol/L sulfuric acid aqueous solution;

Flow rate: 0.6ml/min;

Detection wavelength: 210nm;

Column temperature: 40℃;

Injection volume: 20μl

The chromatogram of the standard solution of the test sample is shown in Figure 12. It can be seen from Figure 12 that the organic acid retention is weak and the peak time is early, which causes matrix interference and cannot achieve effective separation.

Embodiment 4 Determination of formic acid residues in Zolpidem tartrate crude drug

The method in Example 1 was configured to inject the standard solution of the test sample into the chromatographic system, and the chromatogram was recorded.

Chromatography system:

Instrument: Waters e2695 liquid chromatography system;

Chromatographic column: Rezex ROA-Organic Acid H+ (8%), 300×7.8mm;

Mobile phase: 9mmol/L sulfuric acid aqueous solution;

Flow rate: 0.6ml/min;

Detection wavelength: 210nm;

Column temperature: 40℃;

Injection volume: 20μl

The chromatogram of the standard solution of the test sample is shown in Figure 13. It can be seen from Figure 13 that the test sample has a large concentration and a high response, which is easy to interfere with the detection of organic acids.

Embodiment 5 Detection of acetic acid residues in isosorbide mononitrate crude drug

The organic acid reference substance mixed solution was prepared according to the method of Example 1.

Test solution: Precisely weigh 100.27 mg of isosorbide mononitrate and dilute to 10 ml with mobile phase.

The above-mentioned mixed solution of each reference substance and the test solution were injected into the chromatographic system respectively, the chromatogram was recorded, and the acetic acid residual content in the isosorbide mononitrate test sample was calculated by the peak area according to the external standard method.

Instrument: Ultimate 3000 liquid chromatography system;

Chromatographic column: YMC Triart C18, 4.6x150mm, 5μm series Rezex ROA-Organic Acid H+ (8%), 300×7.8mm;

Mobile phase: 1mmol/L sulfuric acid aqueous solution;

Flow rate: 0.4ml/min;

Detection wavelength: 210nm;

Column temperature: 40℃;

Injection volume: 20μL

The spectrum of the mixed reference substance is shown in Figure 14, and the spectrum of the test product is shown in Figure 15. As can be seen from Figure 14, the chromatographic peaks of each organic acid can be effectively separated and the separation is good; it can be seen from Figure 15 that the isosorbide mononitrate API There is no acetic acid residue in it.

Example 6 Detection of acetic acid residues in gimeracil bulk drug

The reference substance mixed solution was prepared according to the method of Example 1.

Test solution: Precisely weigh 100.13 mg of gimeracil in a 10 ml volumetric flask, dilute with mobile phase and dilute to volume.

Inject the above-mentioned reference substance positioning solution, test solution, and test solution adding standard solution into the chromatographic system respectively, record the chromatogram, and qualitatively determine by the retention time of the organic acid in the mixed solution of the reference substance, and calculate the Jimei by the peak area according to the external standard method. Residual content of acetic acid in the pyrimidine test product.

Instrument: Ultimate 3000 liquid chromatography system;

Chromatographic column: Welch XB-C18, 150×4.6mm, 5μm series Aminex HPX-87H (8%), 300×7.8mm;

Mobile phase: 5mmol/L phosphoric acid aqueous solution;

Flow rate: 0.8ml/min;

Detection wavelength: 210nm;

Column temperature: 40℃;

Injection volume: 20μl

The spectrum of the mixed reference substance is shown in Figure 16, and the spectrum of the test product is shown in Figure 17. It can be seen from Figure 16 that the chromatographic peaks of each organic acid can be effectively separated and the resolution is good; it can be seen from Figure 17 that the gimeracil API does not contain Acetic acid remains.

Embodiment 7 Determination of acetic acid residues in riluzole bulk drug

The reference substance mixed solution was prepared according to the method of Example 1.

Test solution: Precisely weigh 100.13 mg of riluzole into a 10 ml volumetric flask, dilute with mobile phase and make up to volume.

Inject the above-mentioned reference substance positioning solution, test solution, and test sample addition solution into the chromatographic system respectively, record the chromatogram, and qualitatively determine by the retention time of the organic acid in the mixed solution of the reference substance, and calculate the interest rate by the peak area according to the external standard method. Residual content of acetic acid in the test product of luzole.

Instrument: Waters e2695 liquid chromatography system;

Chromatographic column: phenomenex onyx monolitnic HD-C18, 100×4.6mm series Rezex ROA-Organic Acid H+ (8%), 300×7.8mm;

Mobile phase: 8mmol/L sulfuric acid aqueous solution;

Flow rate: 0.5ml/min;

Detection wavelength: 210nm;

Column temperature: 40℃;

Injection volume: 20μl

The spectrum of the mixed reference substance is shown in Figure 18, and the spectrum of the test sample is shown in Figure 19. It can be seen from Figure 18 that the chromatographic peaks of each organic acid can be effectively separated, and the resolution is good; it can be seen from Figure 19 that there is no Contains acetic acid residues.

Example 8 Verification Example

(1) System precision detection

The preparation method of the zolpidem tartrate standard solution for the test sample is the same as that in Example 1. The standard solution for the test product is used as the system suitability solution, and 20 μl of the system suitability solution is precisely measured, injected into the liquid chromatograph, and injected continuously for 6 needles. , measure according to the chromatographic conditions of Example 1, record the chromatogram, calculate the RSD of the organic acid peak area of each chromatogram respectively, and the formic acid retention time and peak area results are shown in Table 1.

Table 1

It can be seen from Table 1 that the RSD value of each organic acid peak area is less than 1%, which shows that the system has good precision.

(2) Linear detection of organic acids

The preparation method of the mixed reference substance stock solution is the same as that in Example 1. The precise amounts are respectively, and 500 μl, 800 μl, 1ml, 1.2ml, 1.5ml and 2ml of the mixed reference substance stock solution are placed in a 10ml measuring bottle respectively, and the mobile phase is added to dilute to the mark. Formulated into fumaric acid 5μg/L, 8μg/L, 10μg/L, 12μg/L, 15μg/L, 20μg/L; formic acid, acetic acid, malonic acid, tartaric acid, citric acid five organic acids 25μg/L, 40μg/L L, 50μg/L, 60μg/L, 75μg/L, 100μg/L linear solutions. Precisely measure 20 μl of each linear solution, inject it into a liquid chromatograph respectively, detect according to the chromatographic conditions of Example 1, record the chromatogram, take the concentration mg/ml as the abscissa, and take the peak area (mAu*min) as the ordinate to carry out Linear regression, the linear regression equation of each organic acid is obtained as shown in Table 2.

Table 2 Linearity test results of organic acids

From the results in Table 2, it can be seen that the concentration range of fumaric acid is 0.005mg/ml ~ 0.02mg/ml, and other various organic acids are in the range of 0.025mg/ml ~ 0.1mg/ml, and the peak area of each component has a good linear relationship with the concentration. .

(3) Sensitivity detection of organic acids

The preparation method of the organic acid reference stock solution is the same as that in Example 1. 500 μl of the fumaric acid stock solution is precisely measured in a 10 ml volumetric flask, diluted with mobile phase and made to volume, as the fumaric acid mother solution. Take 100 μl of tartaric acid stock solution, 200 μl of citric acid stock solution, 200 μl of malonic acid stock solution, 250 μl of formic acid stock solution, 500 μl of acetic acid stock solution and 200 μl of fumaric acid stock solution in a 10 ml volumetric flask, dilute with mobile phase and make up to volume, as the sensitivity solution. Precisely measure 20 μl of the sensitivity solution, inject it into a liquid chromatograph, perform detection according to the chromatographic conditions of Example 1, and record the spectrum. The chromatogram of the sensitivity solution is shown in Figure 20. At this time, the S/N of various organic acids are all about 15, indicating that the detection method of the present invention has high sensitivity.

(4) Accuracy detection of the content of organic acids

The preparation method of the reference substance stock solution, the test substance solution and the mixed reference substance solution is the same as that in Example 1.

50% accuracy test solution: Accurately weigh 100.0 mg of Zolpidem tartrate in a 10 ml volumetric flask, add 0.5 ml of reference stock solution, dilute to the mark with mobile phase, and shake to obtain; prepare 3 copies in the same way.

100% accuracy test solution: Accurately weigh 100.1 mg of Zolpidem tartrate in a 10 ml volumetric flask, add 1 ml of the reference stock solution, dilute to the mark with mobile phase, shake well, and prepare 3 copies in the same way.

150% accuracy test solution: Accurately weigh 100.0 mg of Zolpidem tartrate in a 10-ml volumetric flask, add 1.5 ml of reference stock solution, dilute to the mark with mobile phase, shake well, and then prepare 3 copies in the same way.

Precisely measure each 20 μl of the test solution, mixed control solution, 50% accuracy test solution, 100% accuracy test solution, and 150% accuracy test solution, inject them into a liquid chromatograph, and carry out according to the chromatographic conditions of Example 1. Measure, record the chromatogram, and calculate the recovery rate, average recovery rate (n=9) and RSD of each organic acid in the three concentration test solutions by peak area according to the 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 organic acids are all greater than 99%, and the RSD is less than 3%, indicating that the detection method of the present invention has good accuracy.

Claims (10)

1. the detection method of organic acid in a crude drug, is characterized in that, adopts reversed-phase chromatographic column series ion-exclusion chromatographic column, with sulfuric acid, phosphoric acid or p-toluenesulfonic acid aqueous solution as mobile phase, with ultraviolet detector or The high performance liquid chromatograph with diode array detector was used for analysis and detection. 2. The method according to claim 1, wherein the reversed-phase chromatographic 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 according to claim 1, wherein the ion exclusion chromatography column is a crosslinked resin hydrogen ion chromatography column, preferably a crosslinked sulfonated styrene-divinylbenzene hydrogen ion chromatography column. 4. The method according to claim 3, wherein the crosslinking degree of the ion exclusion chromatography column is 5%, 8% or 10%. 5 . The method according to claim 1 , wherein the mobile phase concentration is 0.1-10 mmol/L. 6 . 6 . The method according to claim 1 , wherein the mobile phase is 5-10 mmol/L sulfuric acid aqueous solution. 7 . 7. The method according to claim 1, wherein the flow rate of the mobile phase is 0.4-0.8 ml/min. 8. The method according to claim 1, wherein the temperature of the chromatographic column is 30-60°C. 9. The method according to claim 1, wherein the detection wavelength is 200-210 nm. 10 . The method according to claim 1 , wherein the injection volume is 10-30 μl. 11 .

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