Application of chlorogenic acid and deuterated substance and / or tritiated substance thereof in detection of chain element and isomer thereof

Abstract

The invention relates to application of chlorogenic acid and a deuterated substance and / or a tritiated substance thereof in detection of chain elements and isomers thereof, and belongs to the technical field of compound detection. The invention also provides a method for detecting the deuterated substance and / or tritiated substance of the chlorogenic acid. The content of the deuterated substance and / or tritiated substance of the chlorogenic acid in a test sample and the content of the isomeride of the deuterated substance are / is detected according to an internal standard method by taking the chlorogenic acid and the deuterated substance and / or tritiated substance as standard substances. According to the characteristics of the desmosine and the isomeride thereof in liquid chromatography detection, nine reagents are screened for liquid chromatography detection, and further screening is carried out according to the time difference between the response peak retention time of the reagents and the response peak retention time of the desmosine and the isomeride thereof. And finally, screening to obtain chlorogenic acid which does not interfere with the retention time of response peaks of the desmosine and the isomeride of the desmosine and is moderate in retention time difference as an internal standard substance, and applying the chlorogenic acid to detection of the desmosine and the isomeride of the desmosine.

Description

Technical Field

[0001] The present invention relates to the technical field of compound detection, and in particular to the use of chlorogenic acid and its deuterated and / or tritiated products in detecting desmosin and its isomers. Background Art

[0002] Bovine ligament elastin peptide is a new raw material for health food, in which desmosin and isodesmosin are its characteristic functional ingredients. The existing detection method is to use liquid chromatography, prepare a mixed standard by desmosin and isodesmosin standard products, and use the external standard method for quantitative calculation to calculate the content of desmosin and isodesmosin in the sample. However, the price of desmosin and isodesmosin standard products is high, and the detection cost is extremely high, which greatly increases the production cost of bovine ligament elastin peptide. Therefore, continue to develop a relatively low-cost, high-accuracy, and good-specificity desmosin and isodesmosin detection method. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide the use of chlorogenic acid and its deuterated and / or tritiated products in detecting desmosin and its isomers, and also provide a method for detecting desmosin and its isomers with good specificity, accuracy and precision.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] In a first aspect, the present invention provides the use of chlorogenic acid and its deuterated and / or tritiated products in detecting desmosin and its isomers.

[0006] According to the characteristics of desmosin and its isomers in liquid chromatography detection, the present invention screened 9 reagents for liquid chromatography detection, and further screened according to the difference between the response peak retention time of the reagent and the response peak retention time of desmosin and its isomers. Finally, chlorogenic acid with no interference with the response peak retention time of desmosin and its isomers and a moderate retention time difference was screened out as an internal standard for application in the detection of desmosin and its isomers.

[0007] As a preferred embodiment of the application of the present invention, the isomer of desmosine is isodesmosine.

[0008] In a second aspect, the present invention provides a method for detecting desmosin and its isomers, using chlorogenic acid and its deuterated and / or tritiated products as standard substances to detect the content of desmosin and its isomers in a test sample according to the internal standard method.

[0009] The method of the present invention is to quantify desmosin and its isomers in the sample to be tested by mixing chlorogenic acid and its deuterated and / or tritiated substances as internal standards with the sample to be tested and then conducting liquid chromatography detection. Chlorogenic acid, desmosin and its isomers can be completely separated under the liquid chromatography conditions of the present method, and the retention time difference between desmosin and its isomers and the response peak of chlorogenic acid is moderate, which can reduce the error caused by the instrument, and at the same time reduce the cost of detection, instrument error and technical error. In addition, the detection method of the present invention also has good specificity, accuracy and precision, and is suitable for detecting desmosin and its isomers.

[0010] As a preferred embodiment of the method of the present invention, the internal standard method comprises the following steps:

[0011] A1. Prepare a standard stock solution by mixing chlorogenic acid, deuterated chlorogenic acid and / or tritiated chlorogenic acid with methanol, and add the stock solution to the sample solution to be tested to prepare an internal standard test solution;

[0012] A2, diluting the standard substance mother solution obtained in step A1 to obtain an internal standard reference substance solution;

[0013] A3. Perform high performance liquid chromatography on the internal standard test solution and the internal standard reference solution obtained in steps A1 and A2, and calculate the content of desmosine and its isomers in the test sample based on the response peak area.

[0014] As a preferred embodiment of the method of the present invention, in step A3, the detection conditions of the high performance liquid chromatography are as follows:

[0015] The chromatographic column is a C18 liquid chromatography column;

[0016] The mobile phase consists of mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is a 0.08-0.12 wt % sodium heptane sulfonate aqueous solution;

[0017] The elution conditions were as follows: the volume ratio of mobile phase A to mobile phase B was mobile phase A:mobile phase B=(4-8):(92-96);

[0018] The elution time is 85-100 min;

[0019] The detector wavelength is 270 nm;

[0020] Column temperature is 28-42°C;

[0021] The flow rate is 0.7-1.2mL / min.

[0022] As a preferred embodiment of the method of the present invention, the chromatographic column is Advanced MDK C18-AR, with a specification of 4.6 mm×250 mm and a particle diameter of 5 μm.

[0023] As a preferred embodiment of the method of the present invention, the sodium heptane sulfonate concentration of the mobile phase B is 0.1 wt % and the pH value is 3.28±0.1.

[0024] As a preferred embodiment of the method of the present invention, the elution condition is that the volume ratio of mobile phase A to mobile phase B is mobile phase A:mobile phase B=(5-7):(93-95).

[0025] Within the preferred parameter range, the present invention experimentally confirms that different mobile phase ratios can effectively separate chlorogenic acid, desmosin and their isomers, among which the separation effect of chlorogenic acid, desmosin and their isomers is best when mobile phase A:mobile phase B=5:95.

[0026] As a preferred embodiment of the method of the present invention, the column temperature is 30-40°C.

[0027] In the preferred parameter range, the present invention experimentally confirmed that different column temperatures can effectively separate chlorogenic acid, desmosin and their isomers, and the separation is best when the column temperature is 30°C.

[0028] As a preferred embodiment of the method of the present invention, the flow rate is 0.8-1.0 mL / min.

[0029] Under the preferred parameter range, the present invention experimentally confirms that different flow rates have little effect on the separation of chlorogenic acid, desmosin and their isomers.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The method of the present invention selects chlorogenic acid as an internal standard, whose response peak retention time is close to that of desmosin and its isomers under the same liquid chromatography conditions, from a variety of standard products, and develops a new quantitative method for desmosin and its isomers, which has the advantages of reducing detection costs, instrument errors, and technical errors. At the same time, the detection method of the present invention has good specificity, accuracy and precision, and is suitable for detecting desmosin and its isomers. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 1 is a liquid chromatogram of different standard products in Experimental Example 1 of the present invention;

[0033] Figure 2 It is a liquid chromatogram of different mobile phase flow rates in Experimental Example 2 of the present invention;

[0034] Figure 3 It is the liquid chromatogram of different mobile phase ratios in Experimental Example 2 of the present invention;

[0035] Figure 4 It is the liquid chromatogram of different column temperatures in Experimental Example 2 of the present invention;

[0036] Figure 5 This is a liquid chromatogram showing the specificity of the method in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0037] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0038] Acetonitrile, methanol, and sodium heptane sulfonate were all of chromatographic grade and provided by MacLean;

[0039] The water was ultrapure water;

[0040] The chromatographic column is Advanced MDK C18-AR, with specifications of 5 μm, 4.6 × 250 mm, provided by Avantor;

[0041] The remaining reagents and consumables, unless otherwise specified, can be obtained from commercial sources.

[0042] Experimental Example 1

[0043] An internal standard reference substance suitable for detecting desmosin and isodesmosin was screened from the standard substances with a peak wavelength close to 270nm. The specific scheme is as follows:

[0044] According to the optimal detection wavelengths of existing standard substances puerarin, hesperidin, geniposide, hydroxytyrosol, chlorogenic acid, aucubin, rutin, 5-methoxytryptamine, and rice bran fatty alkyl alcohols in HPLC (Table 1), it can be concluded that puerarin, hesperidin, geniposide, hydroxytyrosol, chlorogenic acid are closest to the optimal detection wavelength (270 nm) of desmosin and isodesmosin, so the above standard substances are selected for the next step of screening.

[0045] Table 1 Optimal detection wavelengths for different standards

[0046] Standards Optimal detection wavelength Puerarin 250nm Hesperidin 285nm Geniposidic acid 240nm Hydroxytyrosol 275nm Chlorogenic acid 320nm Aucubin 203nm Rutin 360nm 5-Methoxytryptamine 222nm Rice bran fatty alcohol 200nm

[0047] According to Table 2, standard substances other than desmosin and isodesmosin were weighed to prepare methanol solutions. Desmosin and isodesmosin were respectively used as solvents with 0.02 mol / L hydrochloric acid solution, and mixed in a volume ratio of desmosin: isodesmosin = 1:1 to prepare a mixed standard solution. The test was performed using an Agilent 1260 liquid chromatograph. The test results are shown in Figure 1 , the liquid chromatography conditions are as follows:

[0048] Mobile phase A was acetonitrile, mobile phase B was 0.1 wt % sodium heptane sulfonate aqueous solution (pH = 3.28 ± 1), and the volume ratio of mobile phase A to mobile phase B was A: B = 5:95;

[0049] The chromatographic column was Advanced MDK C18-AR (4.6 × 250 mm, 5 μm);

[0050] The flow rate is 0.8 mL / min;

[0051] The detection wavelength is 270nm;

[0052] The injection volume was 10 μL;

[0053] The column temperature was 30°C.

[0054] Table 2 Preparation of different standard solutions

[0055]

[0056] like Figure 1 As shown in the figure, the retention time of puerarin, geniposide and hydroxytyrosol is quite different from that of desmosin and isodesmosin, which may lead to reduced sensitivity due to the instability of the instrument; the retention time of hesperidin partially overlaps with that of desmosin and isodesmosin, which may cause interference; the retention time of chlorogenic acid is relatively close to that of desmosin and isodesmosin, and no interfering peaks appear within the retention time of desmosin and isodesmosin, so chlorogenic acid was selected as the internal standard reference substance for desmosin and isodesmosin.

[0057] Experimental Example 2

[0058] The liquid chromatography conditions were further optimized, and the specific scheme is as follows:

[0059] 1. Flow rate optimization.

[0060] According to the liquid chromatography conditions of Experimental Example 1, the flow rate was adjusted to 1.0 mL / min or maintained at 0.8 mL / min, and the other parameters remained unchanged. The test sample was a mixture of chlorogenic acid, desmosin and isodesmosin. The results are shown in Figure 2 , the relative retention times are shown in Table 3.

[0061] The method for preparing a mixed solution of chlorogenic acid, desmosin and isodesmosin is as follows: prepare the chlorogenic acid standard stock solution according to Table 2, take 0.8 mL of the chlorogenic acid standard stock solution, add methanol to make the total volume 10 mL, and obtain a chlorogenic acid standard solution with a chlorogenic acid concentration of 72.4104 μg / mL, mix the chlorogenic acid standard solution with the desmosin and isodesmosin standard solutions prepared according to Table 2 in a volume ratio of 1:1:1 to obtain a mixed solution.

[0062] Table 3 Relative retention time of different treatments

[0063]

[0064] like Figure 2 As shown in Table 3, when the flow rate of the mobile phase was 0.8 mL / min and 1.0 mL / min, the separation of chlorogenic acid, desmosin and isodesmosin was good, and the response peaks were relatively symmetrical, indicating that the mobile phase flow rate of 0.8-1.0 mL / min was suitable for the detection of desmosin and isodesmosin using chlorogenic acid as an internal standard.

[0065] 2. Volume ratio of mobile phase.

[0066] According to the liquid chromatography conditions of Experimental Example 1, the volume ratio of mobile phase A and mobile phase B was set to A:B=5:95 or A:B=7:93, and the other parameters remained unchanged. The test sample was a mixture of chlorogenic acid, desmosin and isodesmosin. The results are shown in Figure 3 , relative retention times are shown in Table 4.

[0067] Table 4 Relative retention time of different treatments

[0068]

[0069] like Figure 3 As shown in Table 4, when the volume ratio of the mobile phase is A:B=5:95 and A:B=7:93, the separation of chlorogenic acid, desmosin and isodesmosin is better, and the response peaks are more symmetrical. When A:B=5:95, the separation of chlorogenic acid, desmosin and isodesmosin is better, and when A:B=7:93, the detection time is shorter. Therefore, the mobile phase with a volume ratio of A:B=(5-7):(93-95) is suitable for the detection of desmosin and isodesmosin using chlorogenic acid as an internal standard.

[0070] 3. Column temperature.

[0071] According to the liquid chromatography conditions of Experimental Example 1, the column temperature was set to 30°C, 35°C and 40°C, and the other parameters were unchanged. The test sample was a mixture of chlorogenic acid, desmosin and isodesmosin. The results are shown in Figure 4 , the relative retention times are shown in Table 5.

[0072] Table 5 Relative retention time of different treatments

[0073]

[0074] like Figure 4 As shown in Table 5, when the column temperature is 30°C, 35°C and 40°C, the separation of chlorogenic acid, desmosin and isodesmosin is good, and the response peaks are relatively symmetrical. Therefore, the column temperature of 30-40°C is suitable for the detection of desmosin and isodesmosin using chlorogenic acid as the internal standard.

[0075] Experimental Example 3

[0076] Methodology verification was performed according to the liquid chromatography conditions of Example 1:

[0077] 1. Detection limit and quantification limit.

[0078] According to the liquid chromatography conditions of Experimental Example 1, the chlorogenic acid reference solution was diluted step by step and calculated by the workstation chromatography software. The injection volume when the signal-to-noise ratio of chlorogenic acid was 10:1 was the quantitative limit of chlorogenic acid. The injection volume when the signal-to-noise ratio of chlorogenic acid was 3:1 was the quantitative limit of chlorogenic acid. The results of the detection limit and quantitative limit are shown in Table 6.

[0079] Table 6 Detection limit and quantification limit of the method

[0080] sample Detection limit (μg / mL) Limit of quantification (μg / mL) Chlorogenic acid 2.24 8.96

[0081] When the chlorogenic acid in the diluent was 8.96 μg / mL, the signal-to-noise ratio of its signal to the baseline noise signal was 10.04, that is, the above concentration was set as the quantitative limit of the method. When the chlorogenic acid in the diluent was 2.24 μg / mL, the signal-to-noise ratio of its signal to the baseline noise signal was 3.08, that is, the above concentration was set as the quantitative limit of the method.

[0082] 2. Method specificity.

[0083] The test solution, internal standard reference solution and blank sample solution were subjected to liquid chromatography according to the chromatographic conditions of Experimental Example 1. The results are shown in Figure 5 .

[0084] Preparation of test solution: accurately weigh 0.1g bovine ligament elastin peptide sample and place it in a hydrolysis tube, add 10mL 6mol / L hydrochloric acid solution, vortex, replace with nitrogen by nitrogen blower for not less than 30s, quickly seal the tube, place it in a 110℃ oven for hydrolysis for 24h, take it out and cool it, transfer it to an evaporating dish, add an appropriate amount of water to rinse the hydrolysis tube, so that the hydrolyzed sample is completely transferred to the evaporating dish, and place the evaporating dish in a boiling water bath to evaporate to near dryness. Rinse the evaporating dish with 0.02mol / L hydrochloric acid solution in small amounts and multiple times, and combine the washing liquid into a 5mL volumetric flask. Use 0.02mol / L hydrochloric acid solution to make up to the scale, then filter, accurately transfer 1mL of filtrate and 0.5mL of chlorogenic acid standard solution (chlorogenic acid concentration is 72.4104μg / mL) into the injection vial, mix well, and obtain the test solution.

[0085] The internal standard chlorogenic acid reference solution was prepared according to the configuration shown in Table 2.

[0086] The blank sample solution was 0.02 mol / L hydrochloric acid solution.

[0087] like Figure 5 As shown, there are multiple impurities in the hydrolyzed bovine ligament elastin, but it is not affected by chlorogenic acid, and the blank solvent does not cause any interfering chromatographic peaks, so the specificity of the method of the present invention meets the requirements.

[0088] 3. Method accuracy.

[0089] The accuracy of this method was investigated by a spike recovery test of the sample. 0.1 g of bovine ligament elastin peptide sample was taken, and the sample was prepared according to the preparation method of the test solution. 0.5 mL of the test solution was accurately pipetted into a 2 mL volumetric flask, and the pipetting was repeated 9 times. Then, different concentrations of desmosin and iso-desmosin reference substance solutions were added to 9 volumetric flasks, and the solution was fixed to the scale with 0.02 mol / L hydrochloric acid solution and mixed evenly. Then the chromatographic conditions in Example 1 were tested and the chromatogram was recorded. The corresponding common content of the sample solution was confirmed by referring to the retention time of the reference solution, and the content of desmosin and iso-desmosin was calculated by peak area. The spike recovery of each reference substance was calculated, and the results obtained were shown in Tables 7 and 8.

[0090] Table 7. Results of the spike recovery experiment of isodesmosine

[0091]

[0092] Table 8 Chain desmosin spike recovery test results

[0093]

[0094]

[0095] As shown in Tables 7 and 8, the recoveries of desmosin and isodesmosin reference substances were between 90% and 110%, meeting the requirements of GB / T27417, indicating that the method has high accuracy and accurate test results.

[0096] 4. Method precision.

[0097] By using the same batch of samples E20230209001, 7 sample solutions were repeatedly prepared according to the preparation method of the internal standard test solution in Example 1, and then the test was performed according to the chromatographic conditions in Example 1, and the chromatogram was recorded. The retention time and ultraviolet spectrum were used for qualitative analysis, and the peak area of ​​chlorogenic acid was used for quantitative analysis, and the concentration of iso-chained chain was calculated by the internal standard method. The peak area of ​​iso-chained chain in the sample was collected, and the concentration of iso-chained chain was calculated by the correction factor control method using chlorogenic acid as the reference substance. The correction factors of chlorogenic acid, iso-chained chain, and chained chain were 3.7833 and 1.8748, respectively. The relative retention times of chlorogenic acid, iso-chained chain, and chained chain were approximately 0.68 and 0.62, respectively. The results are shown in Table 9.

[0098] Table 9 Precision test results

[0099]

[0100]

[0101] As can be seen from Table 9, the RSD values ​​of the contents of desmosin and isodesmosin in the samples were less than 10%, indicating that the method had good repeatability and high precision.

[0102] 5. Method stability.

[0103] According to the preparation method of the internal standard test solution in Example 1 for the same batch of samples E20230209001, 1 sample solution was prepared, stored in the laboratory (24°C, humidity 60%), and then the test was repeated within 4 days according to the chromatographic conditions in Example 1, and the chromatogram was recorded. The retention time and ultraviolet spectrum were used for qualitative analysis, and the peak area of ​​chlorogenic acid was used for quantitative analysis, and the concentration of iso-chain was calculated by the internal standard method. The peak area of ​​iso-chain in the sample was collected, and the concentration of iso-chain was calculated by the correction factor control method using chlorogenic acid as the reference substance. The correction factors of chlorogenic acid, iso-chain and chain were 3.7833 and 1.8748, respectively. The relative retention times of chlorogenic acid, iso-chain and chain were approximately 0.68 and 0.62, respectively. The results are shown in Table 10.

[0104] Table 10 Stability test results

[0105]

[0106] As can be seen from Table 10, the RSD values ​​of desmosin and isodesmosin contents in samples from day 0 to day 2 were less than 1.3%, indicating that the method had good stability within 2 days.

[0107] Experimental Example 4

[0108] The external standard method was compared with the internal standard method of the present invention.

[0109] The samples of batch E20230209001 were tested according to the liquid chromatography conditions of Experimental Example 1, and desmosin and isodesmosin in the tested samples were quantified according to the external standard method and the internal standard method.

[0110] The test sample preparation method for batch E20230209001 was the same as the test sample solution preparation method in Experimental Example 3, wherein the test sample solution of the external standard method did not contain chlorogenic acid.

[0111] The preparation method of the mixed standard solution of desmosin and isodesmosin is the same as the preparation method of the mixed standard solution in Experimental Example 1.

[0112] The chlorogenic acid standard solution was prepared according to Table 2.

[0113] The specific steps of the external standard method are: subjecting the test solution (excluding chlorogenic acid), the mixed standard solution of desmosin and isodesmosin to liquid chromatography detection, confirming the corresponding common content of the test solution with reference to the retention time of the mixed standard solution of desmosin and isodesmosin, and calculating the content of desmosin and isodesmosin by peak area.

[0114] The contents of isodesmosin and desmosin in the sample are calculated using chromatography data processing software or according to formula (1): (the calculation result is retained to two significant figures):

[0115]

[0116] Where: X 1 - the content of isodesmosin or desmosin in the sample (%);

[0117] C s —The concentration of isodesmosin or desmosin in the control solution (μg / mL);

[0118] A 1 - Peak area of ​​isodesmosin or desmosin in the sample;

[0119] V—sample fixed volume (mL);

[0120] A s - Peak area of ​​isodesmosin or desmosin in standard solution;

[0121] m—sample mass, (g).

[0122] The specific steps of the internal standard method are: subject the test solution and the chlorogenic acid standard solution to liquid chromatography detection, qualitatively determined by retention time and ultraviolet spectrum, quantitatively determined by the peak area of ​​chlorogenic acid, and calculating its isodesmosin concentration by the internal standard method. The peak area of ​​isodesmosin in the sample is collected, and the isodesmosin concentration is calculated by the correction factor control method using chlorogenic acid as a reference substance. The correction factors of chlorogenic acid, isodesmosin, and desmosin are 3.7833 and 1.8748, respectively. The relative retention times of chlorogenic acid, isodesmosin, and desmosin are approximately 1.92 and 2.13, respectively.

[0123] The quantitative results of the external standard method are shown in Table 11, and the quantitative results of the internal standard method are shown in Table 12.

[0124] Table 11 Quantitative results of external standard method

[0125]

[0126] Table 12 Quantitative results of internal standard method

[0127]

[0128] As shown in Tables 11 and 12, by comparing the test results of the external standard method and the internal standard method, it can be found that the test results of desmosin and isodesmosin content are not much different, which leads to the conclusion that the internal standard method is feasible.

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Application of chlorogenic acid and its deuterated and / or tritiated derivatives in the detection of desmosin and its isomers.

2. The use according to claim 1, characterized in that The isomer of desmosine is isodesmosine.

3. A method for detecting desmosine and its isomers, characterized in that: Chlorogenic acid and its deuterated and / or tritiated products were used as standard substances to detect the contents of desmosin and its isomers in the test samples according to the internal standard method.

4. The method according to claim 3, characterized in that The internal standard method comprises the following steps: A1. Prepare a standard stock solution by mixing chlorogenic acid, deuterated chlorogenic acid and / or tritiated chlorogenic acid with methanol, and add the stock solution to the sample solution to be tested to prepare an internal standard test solution; A2, diluting the standard substance mother solution obtained in step A1 to obtain an internal standard reference substance solution; A3. Perform high performance liquid chromatography on the internal standard test solution and the internal standard reference solution obtained in steps A1 and A2, and calculate the content of desmosine and its isomers in the test sample based on the response peak area.

5. The method according to claim 4, characterized in that In step A3, the detection conditions of the high performance liquid chromatography are as follows: The chromatographic column is a C18 liquid chromatography column; The mobile phase consists of mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is a 0.08-0.12 wt % sodium heptane sulfonate aqueous solution; The elution conditions were as follows: the volume ratio of mobile phase A to mobile phase B was mobile phase A:mobile phase B=(4-8):(92-96); The elution time is 85-100 min; The detector wavelength is 270 nm; Column temperature is 28-42°C; Flow rate 0.7-1.2 mL / min; The injection volume was 8-10 μL.

6. The method according to claim 5, characterized in that The chromatographic column is Advanced MDK C18-AR, with a specification of 4.6 mm×250 mm and a particle diameter of 5 μm.

7. The method according to claim 5, characterized in that The sodium heptane sulfonate concentration of the mobile phase B is 0.1 wt %, and the pH value is 3.28±0.

1.

8. The method according to claim 5, characterized in that The elution condition is that the volume ratio of mobile phase A to mobile phase B is mobile phase A:mobile phase B=(5-7):(93-95).

9. The method according to claim 5, characterized in that The column temperature is 30-40°C.

10. The method according to claim 5, characterized in that The flow rate is 0.8-1.0 mL / min.

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